Effect of peptide-based captopril analogues on angiotensin converting enzyme activity and peroxynitrite-mediated tyrosine nitration

Article abstract of DOI:10.1039/c1ob05148b

Angiotensin converting enzyme (ACE) regulates the blood pressure by converting angiotensin I to angiotensin II and bradykinin to bradykinin 1-7. These two reactions elevate the blood pressure as angiotensin II and bradykinin are vasoconstrictory and vasodilatory hormones, respectively. Therefore, inhibition of ACE is an important strategy for the treatment of hypertension. The natural substrates of ACE, i.e., angiotensin II and bradykinin, contain a Pro-Phe motif near the site of hydrolysis. Therefore, there may be a Pro-Phe binding pocket at the active site of ACE, which may facilitate the substrate binding. In view of this, we have synthesized a series of thiol- and selenol-containing dipeptides and captopril analogues and studied their ACE inhibition activities. This study reveals that both the selenol or thiol moiety and proline residues are essential for ACE inhibition. Although the introduction of a Phe residue to captopril and its selenium analogue considerably reduces the inhibitory effect, there appears to be a Phe binding pocket at the active site of ACE. The Royal Society of Chemistry 2011.

Full text of DOI:10.1039/c1ob05148b

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PAPER
Effect of peptide-based captopril analogues on angiotensin converting enzyme
activity and peroxynitrite-mediated tyrosine nitration†
Bhaskar J. Bhuyan and Govindasamy Mugesh*
Received 27th January 2011, Accepted 12th April 2011
DOI: 10.1039/c1ob05148b
Angiotensin converting enzyme (ACE) regulates the blood pressure by converting angiotensin I to
angiotensin II and bradykinin to bradykinin 1–7. These two reactions elevate the blood pressure as
angiotensin II and bradykinin are vasoconstrictory and vasodilatory hormones, respectively. Therefore,
inhibition of ACE is an important strategy for the treatment of hypertension. The natural substrates of
ACE, i.e., angiotensin II and bradykinin, contain a Pro-Phe motif near the site of hydrolysis. Therefore,
there may be a Pro-Phe binding pocket at the active site of ACE, which may facilitate the substrate
binding. In view of this, we have synthesized a series of thiol- and selenol-containing dipeptides and
captopril analogues and studied their ACE inhibition activities. This study reveals that both the selenol
or thiol moiety and proline residues are essential for ACE inhibition. Although the introduction of a
Phe residue to captopril and its selenium analogue considerably reduces the inhibitory effect, there
appears to be a Phe binding pocket at the active site of ACE.
Introduction
Angiotensin converting enzyme (ACE) catalyzes the production
of angiotensin II (Ang II) from angiotensin I (Ang I)1 and
degradation of bradykinin to bradykinin 1–7,2 which result in
the elevation of blood pressure.3–9 The conversions of both these
substrates are domain dependent as the N-terminal domain
of somatic ACE cleaves the terminal dipeptide of bradykinin
and the C-terminal domain produces Ang II.10,11 The active
Fig. 1 Captopril (1) and its analogues studied as ACE inhibitors.17
site of both these domains contains a zinc(II) ion coordinated
by histidine and glutamate residues. The inhibition of ACE by
the synthesis and ACE inhibition activities of some captopril
analogues having a Phe residue. We also describe the effect of
these captopril analogues on peroxynitrite-mediated nitration of
Ang II and bovine serum albumin.
synthetic compounds is an important strategy for the treatment
of hypertension. Furthermore, it is known that hypertension and
oxidative stress are interrelated. Therefore, antihypertensive drugs
having antioxidant activity are considered to be beneficial for the
treatment of hypertension.12–16 Recently, we have shown that the
selenium analogue of captopril (2) and dipeptides 3 and 4 (Fig.
1) not only inhibit ACE but also scavenge peroxynitrite (a strong
oxidant found in vivo).17 In this particular study, we have used
the dipeptides having the terminal acid groups in their protected
form. Therefore, it was thought worthwhile to synthesize the L-Sec-
L-Pro- and L-Cys-L-Pro-based dipeptides (5 and 6, Fig. 2) having
free terminal carboxylate groups. Furthermore, the structures of
Ang I and bradykinin show the presence of a Pro-Phe motif near
the site of hydrolysis, indicating that there may be a specific Phe
binding pocket at the active site of ACE. In this paper, we report
Synthesis of dipeptides and captopril analogues
The Sec-Pro and Cys-Pro dipeptides 3–6 were synthesized by
following a procedure reported by our group.17 The captopril
analogues having an additional amino group 7–14 (Fig. 2) were
synthesized by following a similar procedure. Due to the presence
of reactive thiol and selenol moieties, the protection of these groups
was found to be important in the synthesis of Cys- and Sec-
containing peptides. Particularly, the synthesis of Sec-containing
peptides was difficult due to a facile oxidation of the selenol to the
corresponding diselenide. Therefore, the deprotection of acid and
amino groups was carried out in the diselenide or disulfide form
(Scheme 1). The selenol 5 and thiol 6 required for the inhibition
studies were obtained by reducing the corresponding diselenide
and disulfide, respectively.
Department of Inorganic and Physical Chemistry, Indian Institute of Science,
Bangalore 560 012, India. E-mail: mugesh@ipc.iisc.ernet.in; Fax: +91-80-
2360 1552/2360 0683
† Electronic supplementary information (ESI) available: HPLC data, Mass
and NMR spectra. See DOI: 10.1039/c1ob05148b
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in compound 22 by LiOH afforded 23, which upon reduction
by NaBH4 generated the thiol 7. Similarly, the reduction of
compound 22 by NaBH4 produced compound 8. As the captopril-
based diselenides are generally less stable than the disulfides, a
modified procedure was followed for the synthesis of compounds
9–14 (Scheme 3). According to this procedure, the PMB-protected
selenium compound 24 was coupled with L-Phe-OMe in the
presence of DCC to produce compound 25. Cleavage of the
PMB group by iodine then produced the corresponding diselenide
(28), which can be converted to the corresponding selenols 9
and 10 either after or before the hydrolysis of the methyl ester,
respectively. Compounds 11–14 having L-Tyr and L-Val residues
were synthesized by following a similar method. It should be noted
that the nature of the amino acid residue does not appear to affect
the stability of the final compounds. The 77Se NMR spectra of
the selenium compounds show that the nature of the amino acid
residue does not have any significant effect on the chemical shift
values. However, the introduction of Tyr residue enhances the
solubility of the diselenides and selenols in buffer.
Fig. 2 Selenium analogues of captopril and related derivatives (5–14).
Scheme 1 Synthesis of Sec-Pro and Cys-Pro dipeptides.
The Phe-substituted captopril analogues 7 and 8 were syn-
thesized from captopril as shown in Scheme 2. Although the
coupling of Phe to captopril can be achieved after the protection
of the thiol group of captopril, we found it convenient to oxidize
captopril to the corresponding disulfide (21) before coupling
with Phe. The DCC-mediated coupling of 21 with 3 equiv. of
L-Phe-OMe afforded compound 22. The cleavage of ester group
Scheme 3 Synthesis of Se-captopril having an additional amino acid
residue to the proline moiety. aamino acid: L-Phe, L-Tyr and L-Val.
Angiotensin converting enzyme assay
The conversion of Ang I to Ang II by ACE was studied in the
presence of various inhibitors (Scheme 4). The decrease in the
concentration of Ang II with an increase in the concentration of
inhibitors was followed by HPLC. The peak area corresponding
to Ang II was obtained and, wherever possible, the IC50 values
(concentration of inhibitors required to inhibit 50% of the enzyme
activity) were determined. The reaction mixture was incubated
at 37 ◦C for 30 min prior to analysis. The thiols (6, 7 and 8)
and selenols (5, 9–14) were freshly prepared by reducing the
corresponding disulfides and diselenides by NaBH4 prior to use
and were kept under N2 atmosphere during the assay. The IC50
Scheme 2 Synthesis of captopril having an additional amino acid residue
attached to the proline moiety.
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Table 1 IC50 values for the inhibition of ACE by compounds 1–14
the presence of an L-Phe residue at the C-terminal of the L-Pro
moiety slightly stabilizes the enzyme–inhibitor complex, which
may be due to the presence of a phenylalanine binding pocket
at the enzyme active site. In contrast to the thiols and selenols,
the corresponding disulfides and diselenides exhibited very weak
inhibition. In all the cases, the IC50 values could not be determined
up to 50 mM concentration of the inhibitors. The maximum
inhibition observed at this concentration for the disulfides and
diselenides is given in Table S1 (ESI†). In these experiments,
compound 28 having Phe residues exhibited the highest activity.
These observations indicate that a thiol or selenol moiety in
addition to the Phe-Pro motif is important for the inhibition of
ACE.
Compound
IC50/nM
Compound
IC50/nM
1
18.1 1.0
342 33
207.1 2.3
696.2 12.8
76.2 1.0
5350 435
5150 330
2
36.4 1.5
6480 640
2290 160
40 000a
3
4
5
6
8
10
12
14
7
9
4100 330
11
13
40 000b
40 000a
a 35% enzyme activity was inhibited at 40 000 nM inhibitor concentration.
b 40% enzyme activity was inhibited at 40 000 nM inhibitor concentration.
Assay conditions: the reaction was carried out in HEPES-HCl buffer
(50 mM, pH 8.3) at 37 ◦C with a final concentration of 50 mM Ang I,
60 mM NaCl and 2 milliunits of ACE in 400 mL reaction mixture.
From the X-ray crystal structure, it is known that captopril binds
to the active site of ACE through the thiol moiety. The ACE–
captopril complex is stabilized by additional hydrogen bonding
interactions with the carbonyl group of captopril through His353
˚
˚
(2.54 A) and His 513 (2.69 A) residues. The phenolic –OH group
of Tyr520 interacts with one of the carboxylate oxygens of the
proline moiety.9,19 Furthermore, it is observed that the phenolic
–OH of Tyr520 and t-nitrogen of His353 are positioned in close
˚
proximity to the carboxylate residue of the proline moiety (2.66 A
Scheme 4 Inhibition of ACE-catalyzed conversion of Ang I to Ang II by
the selenium analogues of captopril.
˚
and 3.74 A, respectively, Fig. 3). The weak inhibition of captopril
analogues having an additional amino acid residue may be due
to the steric hindrance at the active site. The activity of the Se-
captopril derivative having an L-Phe residue (compound 9) is about
150 times higher than that of compounds 11 and 13 having L-Tyr
and L-Val residues, respectively. On the other hand, only about
two times decrease in the ACE inhibition activity was observed
when an L-Phe residue was introduced to Se-captopril (2). These
observations suggest that there is a Phe binding pocket at the active
site of ACE, which may stabilize the captopril derivatives having
an L-Phe residue. However, it appears that the interaction of the
carboxylic group of Se-captoril (2) with the enzyme active site is
more important for the inhibition than the interaction of the Phe
residue. Recently, Akif et al.20 have reported the crystal structure
of AnCE (a homologue of ACE) in complex with RXP380, a C-
terminal specific inhibitor of ACE containing a L-Trp residue.
It has been shown that the aromatic group of L-Trp interacts
values obtained for the inhibition of ACE-catalyzed conversion
of Ang I to Ang II by the captopril analogues are summarized in
Table 1. The IC50 values for ACE inhibition by compounds 1–4
are included for comparison.17
From the IC50 values, it is clear that the Sec-Pro dipeptides are
better inhibitors of ACE than the Cys-Pro dipeptides. The free
carboxylate moiety at the C-terminal appears to be important as
the Sec-Pro dipeptide 5 having a free carboxylate moiety inhibits
ACE with an IC50 value of 207.1 2.3 nM, which is about 1.5 times
less than that observed for compound 3 (IC50: 342 33 nM) having
the terminal carboxylate moiety in its ester form. Similarly, the IC50
value for compound 6 (IC50: 2290 160 nM) is about three times
less than that of compound 4 (IC50: 6480 640 nM). The ACE
inhibitory potency of compound 7 (IC50: 696.2 12.8 nM) having
an L-Phe residue is much lower than that of captopril (IC50: 18.1
1.0 nM). However, only a marginal decrease in the activity was
observed when a phenylalanine residue was added to the proline
moiety of Se-captopril. Interestingly, the decrease in the activity
upon introduction of L-Phe is less pronounced in Se-captopril
(2) as compared to that of captopril (1). This is probably due to
the higher reactivity of selenol toward zinc(II) ion as compared
to thiol.18 The IC50 value observed for compound 9 (IC50: 76.2
1.0 nM) is only two times higher than that of Se-captopril (IC50:
36.4 1.5 nM). In contrast, the introduction of other amino acids
such as L-Tyr or L-Val instead of L-Phe drastically reduced the
inhibition activity. For example, the IC50 values for compounds
11 and 13 were found to be ~150 times higher than that of
Se-captopril. These observations indicate that the presence of a
free carboxylic acid at the terminal amino acid is important for
ACE inhibition as compounds 8, 10, 12 and 14 having a methyl
ester group exhibited very weak inhibition. This is similar to the
inhibition properties of Cys-Pro and Sec-Pro peptides for which
stronger inhibition was observed for compounds having a free
carboxylic group. Introduction of an amino acid residue decreases
the ACE inhibition potency of the captopril analogues. However,
Fig. 3 Crystal structure of captopril binding to the active site of ACE.
Captopril binds to ACE through thiolate coordination to Zn(II) and there
are additional hydrogen bonding interactions to carbonyl and carboxylate
groups of captopril through various amino acid residues present at the
active site.9,19
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Table 2 IC50 values for the inhibition of ACE by compounds 5–14, and
with the subsite having aromatic residues such as Phe363. In
testicular ACE, these positions are occupied by Val379 and Val380,
which may be involved in interactions with the L-Phe residue in
compound 9 (Fig. 4). However, these interactions may not be
sufficient to stabilize the ACE-9 complex as the size of L-Phe is
significantly smaller than that of the L-Trp residue.
their corresponding diselenides and disulfides
Compound
IC50/nM
Compound
IC50/nM
5
4.5 0.2
15.2 0.6
14.1 1.0
21.9 0.6
6.0 0.5
6.2 0.4
5.6 0.5
7.2 0.6
6.4 0.3
4.5 0.1
19
20
23
22
31
28
32
29
33
30
3.4 1.5
100a
6
7
45.4 1.5
40.8 1.4
3.6 0.1
2.9 0.1
5.8 0.4
4.0 0.1
3.4 0.2
2.0 0.1
8
9
10
11
12
13
14
a Only 30% of the activity was inhibited at this concentration. Assay
conditions: reactions were carried out in sodium phosphate buffer
(100 mM, pH 7.5) at 22 ◦C with a final concentration of 20 mM Ang
II, 300 mM PN and different concentrations of the inhibitors.
in the concentrations of inhibitor was followed by measuring
the peak area at 215 nm. The concentrations of test compounds
required for the inhibition of 50% of nitration are represented as
IC50 values. The IC50 values obtained for the inhibition by different
sulfur and selenium compounds are summarized in Table 2.
In contrast to the ACE inhibition, the PN-scavenging activity
does not depend significantly on the nature of the amino acid side
chain. The dipeptides 5 and 6 and the corresponding diselenides
and disulfides having additional amino acid residues exhibit
similar effects on the nitration reactions. As expected, the PN-
scavenging activity of all the selenium compounds was found
to be much higher than that of the sulfur analogues. Most of
the diselenides exhibited better activity than the corresponding
selenols. This is in contrast to the captopril analogues in which the
selenols were found to be more active than the corresponding
diselenides.17 It should be noted that the thiols 7 and 8 were
found to be much better scavengers of PN than the corresponding
disulfides 23 and 22, respectively. This is in agreement with
our previous report that the antioxidant activity of captopril is
significantly higher than that of the corresponding disulfide.
It is known that PN inactivates several proteins by tyrosine
nitrations. PN can effectively nitrate one or more tyrosyl residues in
bovine serum albumin (BSA) depending on the PN concentration.
Therefore, we have studied the effect of diselenides and disulfides
on PN-mediated nitration of BSA by immunoblotting experi-
ments. In a typical experiment, 100 mM BSA was incubated with
1.2 mM PN and 60 mM of inhibitor and the reaction was incubated
at 20 ◦C for 30 min. At this concentration of the inhibitors, ~20–
50% inhibition of tyrosine nitration was observed (Fig. 5). Similar
to the inhibition of Ang II, the selenium compounds (19, 28–33)
exhibited better inhibition of PN-mediated nitration of BSA as
compared to the sulfur-containing compounds 22 and 23.
Fig. 4 Possible binding of captopril (1) and compound 9 to the ACE
active site. Val379 and Val380 are in close proximity to the proline moiety
of the captopril–ACE complex,19 which may play an important role in
forming a Phe binding pocket.
PN-mediated nitration assays
To understand the effect of additional amino acid in captopril
analogues on the antioxidant activity,21–24 we carried out the
inhibition of peroxynitrite (PN)-mediated nitration of the tyrosine
residues in Ang II and bovine serum albumin (BSA). Briefly, the
nitration of Ang II was studied by an HPLC method (Scheme
5) and BSA was studied by an immunoblotting method. As the
formation of 3,5-dinitro-Ang II was also observed in the reaction,
only the initial 5–10% of the conversion was followed, for which
only a trace amount of the dinitro compound was produced. The
decrease in the concentration of nitro-Ang II with an increase
To understand the mechanism of inhibition of PN-mediated
nitration by the diselenides and disulfides, compounds 22 and 28
were treated with PN in 50 mM phosphate buffer of pH 7.5 and the
reaction products were analyzed by a mass spectrometer. It was
observed that the reaction of the disulfide 22 with PN essentially
produced only one oxidized product (compound 34, Fig. 6).
Although several peaks were observed in the mass spectrum (Fig.
S3, ESI†), all these peaks can be assigned either to the starting
disulfide or to the oxidized product 34, which are complexed with
Scheme 5 Peroxynitrite-mediated nitration of Ang II to mono-nitro-Ang
II.
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to captopril or Se-captopril remarkably reduces the inhibitory
activity. These observations suggest that the proline carboxylate
plays an important role in the ACE inhibition and there may be
a phenylalanine binding pocket at the active site of ACE. The
antioxidant activity of captopril analogues was studied using PN-
mediated nitration of Ang II and BSA. It was observed that the
introduction of an additional amino acid to proline does not affect
the PN-scavenging activity of the selenium compounds. The better
antioxidant activity of the diselenides as compared to the disulfides
is mainly due to the facile oxidation of the selenium center in the
diselenides
Experimental section
Fig. 5 Immunoblots for the inhibition of PN-mediated nitration of BSA.
100 mM of BSA was incubated with 60 mM of inhibitor and 1.2 mM of PN
for 30 min at 20 ◦C and then subjected to gel electrophoresis.
General procedure
Angiotensin converting enzyme (ACE) and captopril (compound
1) were purchased from Sigma-Aldrich Chemical Co. Compounds
2–4, 15–18 were synthesized by known methods.17 Compounds
5–14 were synthesized in situ by reducing the corresponding
disulfides and diselenides with two equivalents of NaBH4 in
methanol. All experiments involving selenols and thiols were
carried out under dry and oxygen-free nitrogen using standard
Schlenk techniques. Column chromatography was performed on
glass columns loaded with silica gel or on an automated flash chro-
matography system (Biotage) by using pre-loaded silica cartridges.
1H (400 MHz), 13C (100.56 MHz), and 77Se (76.29 MHz) NMR
spectra were obtained on a Bruker 400 MHz NMR spectrometer.
Chemical shifts are cited with respect to SiMe4 as internal (1H
and 13C), and Me2Se as external (77Se) standards. Mass spectral
studies were carried out on a Q-TOF micro mass spectrometer or
on a Bruker Daltonics 6000 plus mass spectrometer with ESI-MS
mode analysis.
Fig. 6 Proposed oxidation products from the reaction of 22 and 28 with
PN.
Synthesis of 19
one or two sodium ions under mass conditions. In contrast, the
treatment of diselenide 28 with PN led to a rapid oxidation of the
selenium center to higher oxidation states. Because of the presence
of two selenium centers, the compound can form multiple oxidized
products. Mass spectral analysis of the reaction mixture indicates
the formation of three distinct products (Fig. S4, ESI†). These data
indicate the formation of compounds 35–38 upon treatment of the
diselenide 28 with PN. As the molecular masses of compounds 36
and 37 are identical, it is difficult to assign the peak at m/z 905.1755
to one of these species. However, these observations suggest that
one molecule of diselenide can scavenge up to three molecules of
PN, which may account for the higher activity of the diselenides
as compared to the disulfides.
0.15 g (0.27 mmol) of compound 17 was dissolved in 10 mL ethanol
and lithium hydroxide (17 mg, 0.4 mmol) was added to it. 10 mL of
water was added to the reaction mixture and allowed to stir for 3 h.
Ethanol was evaporated under reduced pressure and the aqueous
layer was lyophilized to remove water. The dipeptide 19 was
purified by reverse phase flash chromatography (water : methanol;
1 : 3). Yield 0.10 g (71%); 1H NMR (D2O) d (ppm): 1.76–1.89 (m,
3H), 2.14–2.21 (m, 1H), 2.91–2.97 (m, 1H), 3.05–3.11 (m, 1H),
3.24–3.29 (dd, 1H), 3.6–3.48 (m 1H), 3.59–3.62 (m, 1H), 4.50–
4.52 (dd, 2H); 13C NMR (D2O) d (ppm): 25.6, 27.7, 28.8, 44.0,
52.8, 55.7, 158.8, 171.7; 77Se NMR (D2O) d (ppm) 305; ESI-MS:
m/z calcd. for C16H26N4O6Se2 [M + H]+ 531.0261, found 530.8870.
Conclusions
Synthesis of 20
This study suggests that the presence of a free carboxylate at
the proline moiety of captopril analogues is important for an
efficient inhibition of angiotensin converting enzyme. While the
introduction of a phenylalanine residue to captopril drastically
reduces the ACE inhibition activity of the parent compound, such
replacement only marginally affects the activity of Se-captopril.
The introduction of tyrosine or valine amino acid residues
This compound was synthesized following a similar method to that
given for compound 19 using compound 18 as starting material.
Yield 79%; 1H NMR (D2O) d (ppm):1.84–1.95 (m, 3H), 2.22–2.26
(m, 1H), 3.14–3.22 (m, 2H), 3.38–3.50 (m, 2H), 4.18–4.21 (m, 2H),
4.42–4.44 (m, 1H); 13C NMR (D2O) d (ppm): 27.5, 31.6, 39.0, 50.5,
53.7, 62.6, 169.3, 178.1; ESI-MS: m/z calcd. for C16H26N4O6S2
[M + H]+ 435.1372, found 434.9422.
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Synthesis of 22
Synthesis of 26
To a solution of 21 (0.14 g, 0.3 mmol) in chloroform, DCC (0.21 g,
1.0 mmol) and HOBt (0.15 g,◦1.0 mmol) were added successively.
The mixture was stirred at 0 C for 20 min. L-Phe-OMe (0.27 g,
1.0 mmol) was added to the above reaction mixture and allowed
to attain the room temperature slowly. The solution was further
stirred for 10 h. The precipitate was filtered and the filtrate was
washed three times each with KHSO4 solution, sodium carbonate
solution and brine. The organic layer was dried over Na2SO4 and
the compound was purified with the help of flash chromatography
(hexane : ethyl acetate, 1 : 3). Yield 0.15 g (62%); 1H NMR (CDCl3)
d (ppm):1.08–1.09 (d, 6H), 1.80–1.85 (m, 2H), 1.92–1.97 (m, 4H),
2.28–2.32 (m, 2H), 2.57–2.59 (m, 2H), 2.98–3.03 (m, 6H), 3.13–
3.18 (m, 2H), 3.45–3.46 (m, 2H), 3.53–3.55 (m, 2H), 3.69 (s, 6H),
4.57–4.59 (d, 2H), 4.75–4.80, (q, 2H), 7.09–7.11 (d, 4H), 7.20–
7.29 (m, 6H); 13C NMR (CDCl3) d (ppm): 17.8, 25.3, 38.2, 38.3,
41.4, 47.8, 52.8, 53.8, 60.2, 127.4, 128.9, 129.7, 136.6, 171.5, 172.4,
175.4; HRMS (ESI mode) calcd. for C38H50N4O8S2 [M + Na]+
777.2968, found 777.2960.
This compound was synthesized following a similar method to
that given for compound 25 by using L-Tyr-OMe instead of L-
1
Phe-OMe. Yield 63%; H NMR (CDCl3) d (ppm): 1.04–1.16 (d,
3H), 1.24–1.28 (m, 2H), 2.29–2.33 (m, 1H), 2.46–2.49 (m, 1H),
2.61–2.65 (m, 1H), 2.82–2.85 (m, 1H), 2.91–2.96 (m, 2H), 3.05–
3.09 (dd, 1H), 3.32–3.34 (m, 2H), 3.70 (s, 3H), 3.75 (s, 2H), 3.81
(s, 3H), 4.57–4.59 (m, 1H), 4.70–4.72 (m, 1H), 6.68–6.70 (d, 2H),
6.82–6.84 (d, 2H), 6.95–6.97 (d, 2H), 7.20–7.22 (d, 2H); 13C NMR
(CDCl3) d (ppm): 18.9, 25.2, 27.3, 28.0, 28.4, 37.5, 39.9, 47.9, 52.8,
54.1, 55.8, 60.3, 114.4, 116.0, 127.2, 130.4, 130.8, 131.9, 156.3,
158.9, 171.9, 172.5, 176.3; 77Se NMR (CDCl3) d (ppm): 247; ESI-
MS: m/z calcd. for C27H34N2O6Se [M + Na]+ 585.1480, found
585.2116.
Synthesis of 27
This compound was synthesized following a similar method to
that given for compound 25 by using L-Val-OMe instead of L-Phe-
OMe. Yield 71%; 1H NMR (CDCl3) d (ppm): 0.86–0.91 (m, 6H),
1.14–1.16 (d, 3H), 1.80–1.83 (m, 1H), 1.96–1.97 (m, 1H), 2.03–
2.15 (m, 2H), 2.48–2.50 (m, 1H), 2.51–2.53 (m, 1H), 2.77–2.80 (m,
1H), 2.87–2.92 (m, 1H), 3.38–3.44 (m, 2H), 3.71 (s, 3H), 3.76 (s,
2H), 3.80 (s, 3H), 4.39–4.43 (m, 1H), 4.69–4.71 (m, 1H), 6.81–
6.84 (d, 2H), 7.20–7.22 (d, 2H); 13C NMR (CDCl3) d (ppm): 18.1,
19.2, 19.5, 25.5, 27.3, 28.0, 31.3, 39.9, 47.8, 52.5, 55.7, 57.8, 59.8,
114.3, 130.3, 131.9, 158.9, 171.6, 172.6, 176.2; 77Se NMR (CDCl3)
d (ppm): 247; ESI-MS: m/z calcd. for C23H34N2O5Se [M + Na]+
521.1531, found 521.1171.
Synthesis of 23
0.10 g (0.13 mmol) of 22 was dissolved in 10 mL ethanol. 14 mg
(0.33 mmol) lithium hydroxide was added to it followed by 10 mL
of water. The mixture was stirred for 3 h. Ethanol was evaporated
under reduced pressure. The aqueous mixture was acidified to a pH
of 2 by adding KHSO4. The compound was then extracted with
ethyl acetate and the organic layer was dried over Na2SO4. The
compound was purified by flash chromatography (hexane : ethyl
acetate, 1 : 5). Yield 0.08 g (83%); 1H NMR (CDCl3) d (ppm):1.05–
1.06 (d, 3H), 1.80–1.82 (m, 3H), 2.10–2.12 (m, 1H), 2.47–2.51 (m,
1H), 2.84–2.95 (m, 3H), 3.09–3.14 (m, 1H), 3.34–3.47 (m, 2H),
4.46–4.47 (m, 1H), 4.67–4.69 (dd, 1H), 7.05–7.16 (m, 5H); 13C
NMR (CDCl3) d (ppm):14.7, 17.6, 21.5, 25.2, 37.8, 41.3, 48.0,
53.8, 60.9, 127.3, 128.8, 129.9, 136.8, 171.8, 174.0, 175.7; ESI-MS:
m/z calcd. for C36H46N4O8S2 [M - H]- 725.2679, found 725.3537.
Synthesis of 28
To a 10 mL methanolic solution of 25 (0.41 g, 0.75 mmol), iodine
(0.23 g, 0.9 mmol) was added. Water (10 mL) was added to
the reaction mixture and stirred for 15 min. To this, 0.5 mL
of hydrazine hydrate was added to destroy the excess iodine.
Methanol was removed under reduced pressure and 20 mL of
1 M KHSO4 solution was added. The compound was extracted
three times with ethyl acetate and purified by flash chromatography
(hexane : ethyl acetate; 1 : 3). Yield 0.27 g (85%); 1H NMR (CDCl3)
d (ppm): 1.11–1.13 (d, 3H), 1.85–1.94 (m, 3H), 2.29–3.01 (m,
1H), 2.81–2.85 (m, 1H), 2.99–3.04 (m, 2H), 3.14–3.23 (m, 2H),
3.46–3.56 (m, 2H), 3.69 (s, 3H), 4.57–4.59 (m, 1H), 4.78–4.79 (d,
1H), 7.10–7.12 (m, 2H), 7.23–7.29 (m, 3H); 13C NMR (CDCl3)
d (ppm):18.4, 25.2, 27.9, 32.8, 38.2, 39.7, 47.8, 52.7, 53.8, 60.1,
127.3, 128.8, 129.6, 136.4, 171.5, 172.3, 175.5; 77Se NMR (CDCl3)
d (ppm): 301; HRMS (ESI-mode) m/z calcd. for C38H50N4O8Se2
[M + Na]+ 873.1857, found 873.1855.
Synthesis of 25
To a solution of 24 (0.50 g, 1.3 mmol) in chloroform, DCC (0.41 g,
2.0 mmol) and HOBt (0.31 g,◦2.0 mmol) were added successively.
The mixture was stirred at 0 C for 20 min. L-Phe-OMe (0.55 g,
2.0 mmol) was added to the above reaction mixture and allowed
to attain the room temperature. The solution was further stirred
for 10 h. Precipitate was filtered and the filtrate was washed three
times each with KHSO4 solution, sodium carbonate solution and
brine. The organic layer was dried over Na2SO4 and the compound
was purified with the help of flash chromatography (hexane : ethyl
acetate, 1 : 3). Yield 0.48 g (68%); 1H NMR (CDCl3) d (ppm): 1.01–
1.03 (d, 3H), 2.35–2.38 (m, 1H), 2.46–2.49 (m, 1H), 2.09–2.12 (m,
1H), 2.81–2.85 (m, 1H), 2.98–3.01 (m, 1H), 3.11–3.19 (m, 3H),
3.29–3.32 (m, 2H), 3.69 (s, 3H), 3.73 (s, 3H), 3.78 (s, 2H), 4.61–
4.62 (d, 1H), 4.75–4.79 (m, 1H), 4.86–4.89 (m, 1H), 6.81–6.82 (dd,
2H), 7.07–7.11 (m, 2H), 7.18–7.29 (m, 5H); 13C NMR (CDCl3)
d (ppm): 17.2, 25.1, 27.3, 28.1, 28.6, 36.9, 40.1, 47.5, 52.9, 54.3,
56.3, 60.1, 114.9, 116.2, 127.3, 128.9, 130.1, 130.9, 132.9, 155.9,
172.3, 172.8, 176.5; 77Se NMR (CDCl3) d (ppm): 246; ESI-MS:
m/z calcd. for C27H34N2O5Se [M - H]- 545.1555, found 545.0529.
Synthesis of 29
This compound was synthesized following a similar method to
that given for compound 28 by using compound 26. Yield 78%;
1H NMR (CDCl3) d (ppm): 1.13–1.14 (d, 3H), 1.89–1.94 (m, 3H),
2.13–2.16 (m, 1H), 2.81–2.84 (m, 1H), 2.95–3.01 (m, 2H), 3.20–
3.23 (m, 1H), 3.52–3.57 (m, 3H), 3.67 (s, 3H), 4.54–4.56 (m, 1H),
4.75–4.77 (m, 1H), 6.69–6.71 (d, 2H), 6.90–6.92 (d, 2H); 13C NMR
(CDCl3) d (ppm): 18.4, 25.3, 28.6, 30.2, 33.0, 39.9, 48.0, 52.9, 53.9,
60.4, 115.9, 127.1, 130.8, 156.4, 172.0, 172.5, 175.6; 77Se NMR
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(CDCl3) d (ppm): 306; ESI-MS: m/z calcd. for C38H50N4O10Se2
ACE assay
[M + Na]+ 905.1755, found 905.3151.
The assay was performed in 400 mL sample vials and an autosam-
pler was used for sample injection. Ang I and AngII were analyzed
by a reverse-phase HPLC method (Princeton C18 column, 4.6 ¥
150 mm, 5 mm) with isocratic elution of 50 : 50 MeOH : 0.1% TFA
in water. In the ACE inhibition assay, we employed a mixture of
50 mM Ang I, 60 mM sodium chloride, 2 milliunits of ACE in
50 mM HEPES-HCl buffer at pH 8.3 with various concentrations
of the inhibitors. The reaction mixture was incubated at 37 ◦C for
30 min prior to injection. Selenols and thiols were freshly prepared
by reducing the diselenides and disulfides by NaBH4 prior to use
and were kept under N2 atmosphere during the assay. Decrease
in the formation of Ang II with an increase in the concentration
of inhibitor was monitored at 215 nm and the % inhibition was
calculated by comparing the peak areas. The inhibition plots were
obtained by using Origin 6.1 software utilizing sigmoidal curve
fitting and these plots were used for the calculation of IC50 values.
Synthesis of 30
This compound was synthesized following a similar method given
for compound 28 by using compound 27. Yield 86%; H NMR
1
(CDCl3) d (ppm): 0.86–0.91 (m, 6H), 1.22–1.24 (d, 3H), 1.83–1.86
(m, 1H), 2.00–2.16 (m, 3H), 2.36–2.39 (m, 1H), 2.83–2.87 (dd,
1H), 3.07–3.9 (m, 1H), 3.23–3.29 (m, 1H), 3.57–3.59 (m, 2H), 3.70
(s, 3H), 4.40–4.43 (m, 1H), 4.66–4.68 (d, 1H); 13C NMR (CDCl3) d
(ppm): 18.1, 18.8, 19.5, 25.5, 27.4, 31.4, 32.7, 39.9, 48.0, 52.6, 57.8,
60.0, 171.6, 172.7, 175.8; 77Se NMR (CDCl3) d (ppm): 305; HRMS
(ESI mode) m/z calcd. for C30H50N4O8Se2 [M + Na]+ 777.1857,
found 777.2000.
Synthesis of 31
Compound 31 was synthesized following a similar method to that
given for compound 23 by using 28 as starting material. Yield 69%;
1H NMR (CDCl3) d (ppm): 1.02–1.04 (d, 3H), 1.81–1.83 (m, 3H),
2.10–2.12 (m, 1H), 2.73–2.75 (d, 1H), 2.89–2.97 (m, 2H), 3.10–
3.15 (m, 2H), 3.36–3.38 (m, 1H), 3.46–3.48 (m, 1H), 4.47–4.49 (d,
1H), 4.67–4.72 (m, 1H), 7.06–7.20 (m, 5H); 13C NMR (CDCl3)
d (ppm): 18.4, 25.2, 28.3, 30.2, 32.7, 37.9, 48.0, 53.8, 60.5, 127.4,
128.9, 129.9, 136.8, 171.9, 174.1, 175.9; 77Se NMR (CDCl3) d
(ppm): 306; HRMS (ESI mode) m/z calcd. for C36H46N4O8Se2
[M + Na]+ 845.1544, found 845.1724.
Nitration of angiotensin II
HPLC experiments were carried out on a Waters-Alliance system
(Milford, MA) consisting of a 2695 separation module, a 2996
photodiode-array detector and a fraction collector. The assays
were performed in 1.8 mL sample vials and a built-in autosampler
was used for sample injection. The Alliance HPLC System
was controlled with EMPOWER software (Waters Corporation,
Milford, MA). The nitration assay of Ang II was analyzed
by reverse phase HPLC method (Princeton C18 column, 4.6 ¥
150 mm, 5 mm) with isocratic elution of 45 : 55 MeOH : 0.1% TFA
in water. In the PN-mediated nitration of Ang II, we employed a
mixture containing Ang II (20 mM) and peroxynitrite (300 mM)
in sodium phosphate buffer (100 mM) of pH 7.5 without and
with increasing concentration of the inhibitor added to the assay
mixture. The reaction mixture was incubated for 5 min before
injection. The formation of nitro-Ang II was monitored at the
wavelength of 215 nm. The inhibition plots were obtained by using
Origin 6.1 software utilizing sigmoidal curve fitting and these plots
were used for the calculation of IC50 values.
Synthesis of 32
This compound was synthesized from compound 29 by following
a similar method to that given compound 23. Yield 71%; 1H NMR
(MeOH-d4) d (ppm): 1.17–1.19 (d, 3H), 1.92–1.95 (m, 3H), 1.99–
2.01 (m, 1H), 2.89–2.96 (m, 2H), 3.02–3.07 (m, 3H), 3.62–3.65
(m, 2H), 4.424.45 (m, 1H), 4.53–4.56 (m, 1H), 6.66–6.72 (m, 2H),
7.03–7.07 (m, 3H); 13C NMR (MeOH-d4) d (ppm): 17.1, 24.7, 29.4,
29.8, 32.6, 36.5, 39.6, 54.4, 60.3, 115.2, 128.1, 130.4, 156.4, 173.2,
173.6, 175.5; 77Se NMR (MeOH-d4) d (ppm): 301; HRMS (ESI
mode) m/z calcd. for C36H46N4O10Se2 [M + Na]+ 877.1442, found
877.2111.
Inhibition of nitration of BSA
For bovine serum albumin (BSA), the nitration was performed by
the addition of PN (1.2 mM) to BSA (0.1 mM) in 0.5 M phosphate
buffer of pH 7.0 at 20 ◦C. After the addition of PN, the final pH
was maintained below 7.5. The reaction mixture was incubated for
30 min at 20 ◦C. The reactions of BSA with PN were performed
in the presence of various inhibitors at 60 mM final concentration.
Upon performing the reactions, the mixture was denatured by
boiling at 100 ◦C for 5 min in the presence of sample loading dye
and subjected to polyacrylamide gel electrophoresis and western
blot analyses.
Synthesis of 33
This compound was synthesized from compound 30 by following
1
a similar method to that given for compound 23. Yield 61%; H
NMR (CDCl3) d (ppm): 0.85–0.90 (m, 6H), 1.14–1.16 (d, 3H),
1.86–1.96 (m, 2H), 2.06–2.20 (m, 3H), 2.76–2.81 (m, 1H), 2.98–
3.00 (m, 1H), 3.14–3.17 (m, 1H), 3.55 (s, 2H), 4.39–4.42 (dd, 1H),
4.60–4.61 (d, 1H); 13C NMR (CDCl3) d (ppm): 17.9, 18.6, 19.6,
25.5, 28.2, 30.2, 31.5, 40.0, 48.2, 57.6, 60.4, 172.2, 174.8, 175.8;
77Se NMR (CDCl3) d (ppm): 304; HRMS (ESI mode) m/z calcd.
for C28H46N4O8Se2 [M + Na]+ 749.1544, found 749.1534.
Electrophoretic analysis
Gel was prepared with 10% polyacrylamide with 6% stacking gel.
The gel was run in the running buffer of pH 8.3 with glycine and
SDS. After separating the proteins, the gel was analyzed by western
blotting. The proteins were transferred to a PVDF membrane and
the non-specific binding sites were blocked by 5% non-fat skimmed
Synthesis of peroxynitrite (PN)
Peroxynitrite was synthesized following a literature procedure.17
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milk in PBST (blocking solution) for 1 h. Then the membrane
was probed with rabbit polyclonal primary antibody against
nitro-tyrosine (1 : 20000 dilutions) in blocking solution for 1 h
followed by incubation with horseradish peroxidase-conjugated
donkey polyclonal anti-rabbit IgG (1 : 20000 dilutions) for another
1 h. The probed membrane was then washed three times with
blocking solution with 0.1% Tween 20 and the immunoreactive
protein was detected by luminol-enhanced chemiluminiscence
(ECL, Amersham).
8 J. Krapcho, C. Turk, D. W. Cushman, J. R. Powell, J. M. DeForrest, E.
R. Spitzmiller, D. S. Karanewsky, M. Duggan, G. Rovnsak, J. Schwartz,
S. Natarajan, J. D. Godfrey, D. E. Ryono, R. Neubeck, K. S. Atwa and
E. W. Petrillo, Jr, J. Med. Chem., 1988, 31, 1148–1160.
9 H. M. Kim, D. R. Shin, O. J. Yoo, H. Lee and J.-O. Lee, FEBS Lett.,
2003, 538, 65–70; K. Brew, Trends Pharmacol. Sci., 2003, 24, 391–394;
H. R. Evans, E. R. Sturrock and K. R. Acharya, Biochemistry, 2004,
43, 8718–8724.
10 R. Igic, T. Nakajima, H. S. J. Yeh, K. Sorrells and E. G. Erdo¨s,
Kininases. Symposium on Kinin Peptides. Fifth Internatl. Congress
Pharmacol., 1972. San Francisco. In Pharmacology and the Future of
Man; ed.: G. H. Acheson, 1973, vol. 5, pp 307–319; L. T. Skeggs,
F. E. Dorer, J. R. Kahn, K. E. Lentz and M. Levin, Experimental
renal hypertension: the discovery of the renin-angiotensin system. In
Biochemical Regulation of Blood; ed. R. Soffer, John Wiley & Sons, Inc.,
Hoboken 1981, pp 3–38.
11 F. Soubrier, F. Alhenc-Gelas, C. Hubert, J. Allegrini, M. John, G.
Tregear and P. Corvol, Proc. Natl. Acad. Sci. U. S. A., 1988, 85, 9386–
9390; E. Jaspard, L. Wei and F. Alhenc-Gelas, J. Biol. Chem., 1993, 268,
9496–9503; A. G. Tzakos, A. S. Galanis, A. Spyroulias, P. Cordopatis,
E. M. Zoupa and I. Gerothanassis, Protein Eng., Des. Sel., 2003, 16,
993–1003.
Acknowledgements
This study was supported by the Department of Science and
Technology (DST), New Delhi. GM acknowledges the DST for
the award of Ramanna and Swarnajayanti fellowships and BJB
thanks the Council of Scientific and Industrial Research, New
Delhi and Indian Institute of Science Bangalore for a research
fellowship.
12 T. Mak, A. M. Freedman, B. F. Dickens and W. Weglicki, Biochem.
Pharmacol., 1990, 40, 2169–2175.
13 X. Liu, R. M. Engelman, J. A. Rousou, G. A. Cordis and D. K. Das,
Cardiovasc. Drugs Ther., 1992, 6, 437–443.
Notes and References
14 S. Rajagopalan, S. Kurz, T. Mu¨nzel, M. Tarpey, B. A. Freeman, K. K.
Griendling and D. G. Harrison, J. Clin. Invest., 1996, 97, 1916–1923.
15 G. Kojda and D. Harrison, Cardiovasc. Res., 1999, 43, 562–571; V.
Turko and F. Murad, Pharmacol. Rev., 2002, 54, 619–634.
16 M. J. Mihm, S. K. Wattanapitayakul, S.-F. Piao, D. G. Hoyt and J. A.
Bauer, Biochem. Pharmacol., 2003, 65, 1189–1197.
17 B. J. Bhuyan and G. Mugesh, Org. Biomol. Chem., 2011, 9, 1356–1365.
18 R. G. Pearson, Coord. Chem. Rev., 1990, 100, 403–425; M. H. Salter,
Jr., J. H. Reibenspies, S. B. Jones and R. D. Hancock, Inorg. Chem.,
2005, 44, 2791–2797.
1 L. T. Skeggs, W. H. Marsh, J. R. Kahn and N. P. Shumway, J. Exp.
Med., 1954, 99, 175–283; L. T. Skeggs, J. R. Kahn and N. P. Shumway,
J. Exp. Med., 1956, 103, 295–299.
2 K. K. Ng and J. R. Vane, Nature, 1968, 218, 144–150; J. R. Vane, J.
Pharmacol., 1969, 35, 209–242.
3 E. G. Erdo¨s and H. Y. T. Yang, Life Sci., 1967, 6, 569–574; H. Y. T.
Yang and E. G. Erdo¨s, Nature, 1967, 215, 1402–1403.
4 D. W. Cushman and M. A. Ondetti, Nat. Med., 1999, 5, 1110–1112 and
references therein.
5 M. A. Ondetti, B. Rubin and D. W. Cushman, Science, 1977, 196, 441–
444; D. W. Cushman, H. S. Cheung, E. F. Sabo and M. A. Ondetti,
Biochemistry, 1977, 16, 5484–5491.
19 R. Natesh, S. L. U. Schwager, E. D. Sturrock and K. R. Acharya,
Nature, 2003, 421, 551–554.
20 M. Akif, D. Georgiadis, A. Mahajan, V. Dive, E. D. Sturrock, R. E.
Isaac and K. R. Acharya, J. Mol. Biol., 2010, 400, 502–517.
21 S. Burney, J. C. Niles, P. C. Dedon and S. R. Tannenbaum, S. R., Chem.
Res. Toxicol., 1999, 12, 513–520.
22 M. S. Wolin, Arterioscler. Thromb. Vasc. Biol., 2000, 20, 1430–1442; S.
A. B. Greenacre and H. Ischiropoloulos, Free Radical Res., 2001, 34,
541–581.
6 A. Patchett, E. Harris, E. W. Tristram, M. J. Wyvratt, M. T. Wu,
D. Taub, E. R. Peterson, T. J. Ikeler, J. tenBroeke, L. G. Payne,
D. L. Ondeyka, E. D. Thorsett, W. J. Greenlee, N. S. Lohr, R. D.
Hoffsommer, H. Joshua, W. V. Ruyle, J. W. Rothrock, S. D. Aster, A.
L. Maycock, F. M. Robinson, R. Hirschmann, C. S. Sweet, E. H. Ulm,
D. M. Gross, T. C. Vassil and C. A. Stone, Nature, 1980, 288, 280–
283.
23 S. N. Savvides, M. Scheiwein, C. C. Bohme, G. E. Arteel, P. A. Karplus,
K. Becker and R. H. Schirmer, J. Biol. Chem., 2002, 277, 2779–2784;
K. P. Bhabak and G. Mugesh, Chem.–Eur. J., 2010, 16, 1175–1185.
24 S. K. Wattanapitayakul, D. M. Weinstein, B. J. Holycross and J. A.
Bauer, FASEB J., 2000, 14, 271–278.
7 E. W. Petrillo, D. W. Cushman, M. E. Duggan, J. E. Karanewsky, M.
A. Ondetti, B. O’ Reilly, G. C. Rovnyak, J. Schwartz, E. R. Spitzmiller
and N.-Y. Wang, in Peptides: Structure and Function. Proceedings of
the Eighth American Peptide Symposium; ed. V. J. Hurby, D. H. Rich,
Pierce Chemical Co. Rockford, 1983 p 541.
5192 | Org. Biomol. Chem., 2011, 9, 5185–5192
This journal is
The Royal Society of Chemistry 2011
©