Antioxidant activity of peptide-based angiotensin converting enzyme inhibitors

Article abstract of DOI:10.1039/c2ob06533a

Angiotensin converting enzyme (ACE) inhibitors are important for the treatment of hypertension as they can decrease the formation of vasopressor hormone angiotensin II (Ang II) and elevate the levels of vasodilating hormone bradykinin. It is observed that bradykinin contains a Ser-Pro-Phe motif near the site of hydrolysis. The selenium analogues of captopril represent a novel class of ACE inhibitors as they also exhibit significant antioxidant activity. In this study, several di- and tripeptides containing selenocysteine and cysteine residues at the N-terminal were synthesized. Hydrolysis of angiotensin I (Ang I) to Ang II by ACE was studied in the presence of these peptides. It is observed that the introduction of l-Phe to Sec-Pro and Cys-Pro peptides significantly increases the ACE inhibitory activity. On the other hand, the introduction of l-Val or l-Ala decreases the inhibitory potency of the parent compounds. The presence of an l-Pro moiety in captopril analogues appears to be important for ACE inhibition as the replacement of l-Pro by l-piperidine 2-carboxylic acid decreases the ACE inhibition. The synthetic peptides were also tested for their ability to scavenge peroxynitrite (PN) and to exhibit glutathione peroxidase (GPx)-like activity. All the selenium-containing peptides exhibited good PN-scavenging and GPx activities. The Royal Society of Chemistry 2012.

Full text of DOI:10.1039/c2ob06533a

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PAPER
Antioxidant activity of peptide-based angiotensin converting enzyme
inhibitors†
Bhaskar J. Bhuyan and Govindasamy Mugesh*
Received 7th September 2011, Accepted 5th January 2012
DOI: 10.1039/c2ob06533a
Angiotensin converting enzyme (ACE) inhibitors are important for the treatment of hypertension as they
can decrease the formation of vasopressor hormone angiotensin II (Ang II) and elevate the levels of
vasodilating hormone bradykinin. It is observed that bradykinin contains a Ser–Pro–Phe motif near the
site of hydrolysis. The selenium analogues of captopril represent a novel class of ACE inhibitors as they
also exhibit significant antioxidant activity. In this study, several di- and tripeptides containing
selenocysteine and cysteine residues at the N-terminal were synthesized. Hydrolysis of angiotensin I
(Ang I) to Ang II by ACE was studied in the presence of these peptides. It is observed that the
introduction of L-Phe to Sec–Pro and Cys–Pro peptides significantly increases the ACE inhibitory activity.
On the other hand, the introduction of L-Val or L-Ala decreases the inhibitory potency of the parent
compounds. The presence of an L-Pro moiety in captopril analogues appears to be important for ACE
inhibition as the replacement of L-Pro by L-piperidine 2-carboxylic acid decreases the ACE inhibition. The
synthetic peptides were also tested for their ability to scavenge peroxynitrite (PN) and to exhibit
glutathione peroxidase (GPx)-like activity. All the selenium-containing peptides exhibited good PN-
scavenging and GPx activities.
analogues is probably due to some minor changes in the orien-
tation of the methyl group of the side chain attached to the Pro
moiety.
Introduction
Angiotensin converting enzyme (ACE) inhibitors are a major
class of antihypertensive drugs.1–3 Inhibition of ACE leads to a
decrease in the blood pressure due to reduced production of
angiotensin II (Ang II), the blood pressure regulating hormone,4
and elevation in the concentration of vasodilating hormone bra-
dykinin5 in the blood stream. There are two isoforms of ACE,
somatic ACE (sACE), a dimeric enzyme having two identical
zinc(II)-containing active sites, and testicular ACE (tACE), a
monomeric enzyme having a similar active site. It is known that
the N-terminal domain of sACE cleaves the terminal dipeptide
of bradykinin, and the C-terminal domain of sACE and tACE
produce Ang II. A structural comparison of Ang I and bradyki-
nin indicates that these two substrates contain a conserved Pro–
Phe linkage. Recently, we have shown that the introduction of an
additional amino acid to captopril analogues (compounds 1–6,
Fig. 1) decreases the inhibition activity of the parent com-
pounds.6,7b Although the active site of ACE includes a Phe
binding pocket in addition to the Pro binding pocket, the
decrease in the activity upon introduction of Phe to captopril
As bradykinin contains an L-serine (L-Ser) residue attached to
the Pro–Phe moiety (Fig. 2), the tripeptides that resemble the
Ser–Pro–Phe motif may exhibit better ACE inhibition by
mimicking the binding at the active site. In view of this, we have
replaced the Ser residue in Ser–Pro–Phe peptide by L-cysteine
(L-Cys) or L-selenocysteine (L-Sec) as this replacement may
provide a suitable ligand for coordination to the zinc(II) ion at
the active site of ACE.8 In this paper, we report on the synthesis
and ACE inhibition activities of peptides 7–13 having Cys or
Sec moieties (Fig. 3).
It is known that ACE inhibitors having antioxidant activity are
beneficial for the treatment of hypertension as hypertension and
oxidative stress are interdependent.9–12 An enhanced cellular
level of Ang II induces the enzyme NADPH oxidase to generate
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: For HPLC data,
mass and NMR spectra. See DOI: 10.1039/c2ob06533a
Fig. 1 Captopril (2) and peptide analogues reported as ACE
inhibitors.6,7b
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Fig. 2 Structure of bradykinin (1–9) indicating the presence of the
Ser–Pro–Phe motif.
Scheme 1 Synthesis of Sec- and Cys-containing tripeptides 7–10.
Fig. 3 Di- and tripeptides used as ACE inhibitors and peroxynitrite
(PN) scavengers.
a superoxide radical anion (O2˙−), which reacts with the endo-
thelial derived relaxing factor (EDRF), nitric oxide radical
(˙NO), to produce peroxynitrite (PN). PN is known to cause cel-
lular damage as it can nitrate free or protein tyrosine residues as
well as cause lipid peroxidation. Recently, we have shown that
the selenium analogues of captopril exhibit both ACE inhibition
and PN-scavenging activities.6,7 To understand the effect of
different amino acids on PN-scavenging activity, we have tested
compounds 7–13 as inhibitors of PN-mediated nitration of Ang
II. In addition to ACE inhibition and PN-scavenging activity, we
have also studied the glutathione peroxidase (GPx) activity of
the synthetic peptides. GPx is a mammalian enzyme that con-
tains a selenocysteine residue at the active site.13 This enzyme
converts hydroperoxides to water or alcohol by using glutathione
(GSH) as a cofactor.14 The chemistry at the active site of GPx
has been studied extensively with the help of various synthetic
organoselenium compounds.15–17 However, it is not known
whether ACE inhibitors can exhibit GPx activity.
Scheme 2 Synthesis of Cys–Val–Phe tripeptide (11).
Results and discussion
disulfide (26–29) in good yield. Removal of the Boc group by
TFA led to the formation of compounds 30–33, which upon
reduction by NaBH4 produced the required selenols and thiols
7–10.
The Cys–Val–Phe–OMe tripeptide (11) lacking a Pro moiety
was synthesized by following a similar procedure (Scheme 2).
TFA-mediated cleavage of the Boc group in compound 34
afforded 35 in nearly quantitative yield. DCC-mediated coupling
of the amino group in this peptide with the carboxylic group of
PMB-protected Cys (21) produced the corresponding tripeptide
The tripeptides 7–10 bearing Sec and Cys residues at the N-
terminal were synthesized by solution phase peptide synthesis.
These compounds were synthesized by using PMB-protected
Sec or Cys as shown in Scheme 1. The initial steps involved the
removal of the Boc group from compounds 14–16 by TFA to
produce compounds 17–19. The DCC-mediated coupling of the
resulting deprotected dipeptides with PMB-protected Sec (20) or
Cys (21) afforded the tripeptides 22–25. Cleavage of the PMB
group by iodine produced the corresponding diselenides and
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Scheme 4 Inhibition of ACE-catalyzed conversion of Ang I to Ang II
by peptides.
Table 1 IC50 values for the inhibition of ACE by compounds 1–13
Compound
IC50 (nM)
Compound
IC50 (nM)
1
2
3
4
5
6
7
36.4 1.5
18.1 1.0
342 33
6480 640
4100 330
40 000a
8
9
10
11
12
13
3500 100
8700 300
12 700 400
50 000b
Scheme 3 Synthesis of dipeptides 12 and 13 starting from L-piper-
idine-2-carboxylic acid methyl ester.
6850 400
40 000a
183.2 10.6
a Only 35% enzyme activity was inhibited at 40 000 nM inhibitor
concentration. b Only 25% enzyme activity was inhibited at 50 000 nM
inhibitor concentration. Assay conditions: The reactions were carried out
in HEPES–HCl buffer (50 mM, pH 8.3) at 37 °C with a final
concentration of 50 μM Ang I, 60 mM NaCl and 2 milliunits of ACE in
400 μL reaction mixture.
36. The usual iodine-mediated removal of the PMB group,
removal of the Boc group by TFA and reduction of the disulfide
linkage by NaBH4 afforded the Cys–Val–Phe–OMe tripeptide
(11). It should be noted that the yields of Cys-based tripeptides
are slightly higher than those of Sec-based compounds. Further-
more, the thiol groups in the cysteinyl-peptides are more stable
in air as compared to the selenol groups in the Sec-based pep-
tides. To understand the importance of the five-membered ring in
Pro-based ACE inhibitors, the Sec- and Cys-containing dipep-
tides 12 and 13 having six-membered rings were synthesized
(Scheme 3). DCC-mediated coupling of L-piperidine-2-car-
boxylic acid methyl ester to the PMB-protected Sec (20) and
Cys (21) afforded the corresponding dipeptides 39 and 40. The
cleavage of the PMB group in 39 and 40 under mild conditions
using iodine led to the formation of diselenides 41 and 42,
respectively. The TFA-mediated removal of the Boc group fol-
lowed by reductive cleavage of the diselenide and disulfide
bonds in 43 and 44 by NaBH4 afforded the corresponding
selenol 12 and thiol 13, respectively, in good yield.
The hydrolysis 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
various inhibitors was followed by HPLC. The peak area corre-
sponding to Ang II was obtained during the initial 5–10% of the
reaction 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 and selenols were freshly
prepared by reducing the corresponding disulfides and disele-
nides by NaBH4 prior to use and were kept under N2 atmosphere
during the assay. The IC50 values obtained for the inhibition of
ACE-catalyzed conversion of Ang I to Ang II by the di- and tri-
peptides are summarized in Table 1. The IC50 values for ACE
inhibition by compounds 1–6 are included for comparison.6,7
From Table 1, it is clear that the introduction of L-Phe to Sec-
Pro and Cys-Pro peptides increases the ACE inhibitory activity.
For example, the IC50 value for Sec–Pro–Phe–OMe (compound
7, IC50: 183.2 10.6 nM) is much lower than that of Sec–Pro–
OMe dipeptide (compound 3 IC50: 342 33 nM). A similar
change in the activity was observed for the corresponding Cys-
containing peptides (compound 8 vs. 4). In contrast, the substi-
tution of L-Phe by L-Val or L-Ala dramatically decreases the
inhibitory potency. The IC50 value for compound 9 having a Val
residue (8700
300 nM) was found to be almost 50 times
higher than that of compound 7 (IC50: 183.2 10.6 nM). Simi-
larly, the IC50 value for compound 10 having an Ala residue
(12 700 400 nM) was found to be almost 70 times higher than
that of 7. Substitution of Pro in compound 8 by Val reduced the
inhibitory activity. When compound 11 was used as inhibitor,
only 25% inhibition of ACE activity was observed even at
50 μM concentration. These observations clearly indicate that
the Pro moiety in captopril analogues is very important for
efficient inhibition of ACE. Furthermore, it is observed that the
substitution of the five-membered ring of Pro by a six-membered
ring of L-piperidine-2-carboxylic acid increases the IC50 value.
For example, the IC50 value observed for compound 12 having a
six-membered ring (6850 400 nM) is ∼20 times higher than
that of compound 3 (342 33 nM). Similarly, when compound
13 was used, only 35% of enzyme activity was inhibited even at
40 μM concentration. In contrast, the Pro-based compound 4
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Scheme 5 Peroxynitrite-mediated nitration of Ang II to mono-nitro
Ang II.
Table 2 IC50 values for inhibition of PN-mediated nitration of Ang II
by compounds 7–13 and their corresponding diselenides and disulfidesa
Compound
IC50 (μM)
Compound
IC50 (μM)
7
8
9
10
11
12
13
7.0 0.1
20.2 1.1
7.7 0.7
9.0 0.8
19.6 0.2
5.5 0.2
14.9 0.8
30
31
32
33
38
43
44
6.1 0.3
33.7 1.8
4.9 0.3
3.7 0.3
32.4 1.6
3.2 0.2
37.7 3.0
Fig. 4 Crystal structure of captopril bound 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 carboxy-
late groups of captopril through various amino acid residues present at
the active site (PDB code: 1UZF).2a A hydrophobic pocket comprising
of three Phe residues (457, 460 and 527) may stabilize the binding of tri-
peptides having Phe residue to ACE active site.
a Assay condition: Reactions were carried out in sodium phosphate
buffer (100 mM, pH 7.5) at 22 °C with a final concentration of 20 μM
Ang II, 300 μM PN and different concentrations of the inhibitors.
residue is present at a position similar to that of the Phe residue
in compound 7, binds to the periphery of subsite S2′ comprising
Val379 and Val380 residues in sACE.18 A close examination of
the active site of ACE reveals that there is a hydrophobic pocket
(Phe457, Phe460 and Phe527) present at a distance of 4–6 Å
from the Pro carboxylate. These Phe residues may stabilize the
tripeptides having Sec–Pro–Phe residues and may form a stable
enzyme–inhibitor complex. However, to the best of our knowl-
edge, the role of these amino acid residues near the active site
has not been studied in detail.
To evaluate the antioxidant activity of di- and tripeptides, we
have studied the effect of these compounds on peroxynitrite-
mediated nitration of Ang II (Scheme 5). 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. During this
period, the mononitro-Ang II compound was produced as the
major product. The IC50 values obtained for the inhibition of
PN-mediated nitration of Ang II are summarized in Table 2.
From the IC50 values, it is evident that the selenium-containing
peptides are much better scavengers of PN than the sulfur ana-
logues. In contrast to the ACE inhibition property, the PN-
scavenging activity does not depend significantly on the C-term-
inal amino acid residue. The dipeptide 12 exhibited the highest
activity (IC50: 5.5 0.2 μM) among all the selenols and thiols.
The IC50 value for the Sec-containing tripeptide 7 (7.0
0.1 μM) is comparable to that of 9 (7.7 0.7 μM) and 10 (9.0
0.8 μM) in which the terminal Phe amino acid residue is replaced
by Val or Ala, respectively. The selenols were found to be good
scavengers of PN, although the IC50 values obtained for these
compounds were about two times higher than those of the corre-
sponding diselenides. In contrast, the thiols were found to be
more efficient than the corresponding disulfides. This is in agree-
ment with our previous report that diselenides can undergo mul-
tiple oxidation after reaction with PN.6,7
inhibited the ACE activity with an IC50 value of 6480
640 nM.
The crystal structure of the ACE–captopril adduct shows that
the thiol group of the inhibitor interacts with the zinc(II) ion to
form a zinc(II)–thiolate complex.2 The inhibitor is anchored to
the central carbonyl group and the Pro carboxylate group by
hydrogen bonding. The central carbonyl group of captopril is
positioned by two strong hydrogen bonds from the two histidine
residues (His353 and His513). The binding of captopril is also
stabilized by hydrogen bonding of the Pro carboxylate group
with neighboring amino acid residues. The key amino acid resi-
dues involved in such hydrogen bonding are Tyr520, Gln281
and Lys511. Introduction of an additional amino acid residue to
captopril may lead to a loss of hydrogen bonding interactions
between the Pro carboxylate and the polar amino acid residues at
the active site. This is probably responsible for the decrease in
the inhibitory activity upon introduction of an additional amino
acid to the Pro moiety (Fig. 4).
Interestingly, the five-membered ring of the Pro residue
appears to be important for a proper orientation of the carboxy-
late moiety. The substitution of the Pro residue by L-piperidine-
2-carboxylic acid drastically decreases the inhibitory potency
(Table 1). On the other hand, introduction of a Phe residue to
Sec–Pro (3) and Cys–Pro (4) dipeptides significantly increases
the inhibitory potency of the parent compounds. This is probably
due to the presence of a free amino group in the Sec or Cys
moiety (compounds 7 and 8), which can stabilize the ACE–
inhibitor complex by hydrogen bonding. As the introduction of
Tyr or Val drastically decreases the inhibitory potency (com-
pounds 9 and 10), it can be concluded that there is a binding
pocket for the Phe residue at the active site of ACE. This is in
agreement with the previous observations.7,18 Akif et al. have
shown that the ACE inhibitor RXP380, in which an L-tryptophan
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Table 3 Initial rate for the GPx-like antioxidant activity by the test
compounds in the glutathione assaya
Compound
Rate (μM min−1
)
Compound
Rate (μM min−1
)
30
32
38
44
46
48
50
138.5 2.7
165.8 3.9
40.8 0.4
46.7 3.0
36.3 2.3
30.8 3.1
22.2 0.2
31
33
43
45
47
49
Ebselen
34.7 2.9
199.6 17.4
161.8 3.4
150.7 0.3
157.6 1.4
106.8 2.6
156.3 6.4
a Assay conditions: Reactions were carried out in sodium phosphate
buffer (100 mM, pH 7.5) at 22 °C with a final concentration of 100 μM
catalyst, 4 mM GSH, 0.4 mM NADPH, 1.7 unit GR mL−1, and 5 mM
H2O2. The initial rates were corrected for the background reaction
between peroxide and thiol.
Scheme 6 Proposed catalytic cycle for reduction of hydrogen peroxide
by GPx. GR reduces glutathione disulfide to glutathione by using
NADPH as cofactor.
Fig. 5 Diselenides and disulfides used for GPx activity.6,7b
It should be noted that the peptides having a Sec or Cys
residue inhibit PN-mediated nitration of Ang II with IC50 values
that are about 10–50 times lower than the concentration of PN in
the reaction medium (300 μM). Recently, we reported that orga-
noselenium compounds can catalytically scavenge PN.19 It has
been shown that PN oxidizes the selenides (R2Se) to the corre-
sponding selenoxides (R2–SevO), which can react with nitrite
ion (NO2−) to produce nitrate (NO3−) with a consumption of
two molecules of PN in one catalytic cycle. Therefore, we have
investigated the reaction of 33 with PN by using mass spectral
techniques. It was observed that both the selenium centers in
compound 33 undergo two electron oxidation (+2 to +4)
immediately after the addition of PN. However, the complete
regeneration of the diselenide was not observed even after 2
days. In the presence of reducing agents such as glutathione
(GSH), the reduction of selenium centers (+4 to +2) was
observed (Fig. S77, ESI†), suggesting that the diselenides may
exhibit a redox cycle in the presence of biological thiols. The
rapid reduction of the oxidized species by GSH prompted us to
investigate the GPx-like antioxidant activity of the peptides as
GPx uses GSH as cofactor to catalytically reduce hydroperoxides
to alcohols or water. The catalytic cycle of GPx involves the
reduction of 1 equiv of peroxide by using two equiv of GSH as
cofactor (Scheme 6).14–17 The GPx-like antioxidant activity of
the Cys- and Sec-containing peptides was determined by using
the classical glutathione reductase (GR) coupled assay. In brief,
a decrease in the concentration of NADPH was followed as a
function of time at 340 nm wavelength as NADPH is used as a
cofactor by GR to reduce glutathione disulfide (GSSG) to gluta-
thione (GSH) as shown in Scheme 6. The initial rates for the
reduction of H2O2 by GSH in the presence of compounds
30–33, 38, 43–50 (Fig. 5) and ebselen were obtained at identical
experimental conditions and are summarized in Table 3.
Scheme 7 Proposed catalytic cycle for reduction of hydrogen peroxide
by the diselenides.
The GPx activity of most of the diselenides is comparable to
that of ebselen, an organoselenium compound used as a standard
in GPx model reactions. The initial rate for the reduction of
H2O2 by compound 33 (199.6 17.4 μM min−1) was found to
be higher than that of ebselen (156.3 6.4 μM min−1). The
nature of the substituent or ring size does not appear to have any
major effect on the GPx activity. For example, the diselenides 43
and 47 having different heterocycles exhibit almost similar GPx
activity. In contrast to the diselenides, the disulfides are very
poor GPx mimics. To understand the mechanism for the
reduction of H2O2 by the selenium compounds, we analyzed the
different species formed in the reactions by using mass spectro-
metric techniques. When compound 33 was treated with GSH
and H2O2, the selenenyl sulfide (53), selenol (51) selenenic acid
(52) and seleninic acid (54) were observed (Scheme 7 and
Fig. S78, S80, ESI†). In this reaction, the formation of GSSG
was also observed. A comparison of the reactivity of 33 toward
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GSH and H2O2 indicates that the reaction with H2O2 is faster
than that with GSH. This observation suggests that the diselenide
may undergo oxidation before reduction by GSH. In the absence
of GSH, compound 33 reacted with H2O2 to produce several oxi-
dized species. Mass spectral analysis of the products indicated
the formation of –Se–(SevO)– and –(SevO)–(SevO)–
species. However, when GSH was added, all these species disap-
peared to produce the selenol, selenenyl sulfide and selenenic–
seleninic acids. The selenol could be trapped by treating the reac-
tion mixture with bromopropionic acid (Fig. S84, ESI†) (Fig. 5).
According to the mechanism shown in Scheme 7, the disele-
nide 33 reacts with H2O2 and GSH to generate the selenol 51,
which upon reaction with 1 equiv of H2O2 generates the selene-
nic acid (52) with the elimination of a water molecule. The sele-
nenic acid then reacts with 1 equiv of GSH to generate the
selenenyl sulfide (53) intermediate. As in the case of GPx, the
selenenyl sulfide reacts with another equiv of GSH to regenerate
the active selenol species. The formation of seleninic acid (54)
as observed in the mass spectrum can be ascribed to the oxi-
dation of compound 52 in the presence of excess peroxide. It
should be noted that the formation of seleninic acid is very
common in model reactions and the GPx enzyme itself produces
such species under oxidative conditions.14,20 However, the sele-
ninic acids can be converted to selenenyl sulfides and then to
selenols by treating with an excess amount of GSH. In biological
systems, where GSH concentration is high, the seleninic acid
may lie off the main catalytic pathway. Compound 33 represents
the first example of a Pro-based peptide that exhibits GPx
activity.21
Experimental section
General procedure
Angiotensin converting enzyme (ACE) and captopril were pur-
chased from Sigma-Aldrich Chemical Co. All experiments invol-
ving selenols and thiols were carried out under dry and oxygen
free nitrogen using standard Schlenk techniques. Column chrom-
atography was performed on glass columns loaded with silica
gel or on an automated flash chromatography 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.
Synthesis of 1422
To a 100 mL solution of Boc-L-Pro (1.07 g, 5 mmol) in chloro-
form, DCC (1.54 g, 7.5 mmol) and HOBt (1.15 g, 7.5 mmol)
were added at 0 °C. The mixture was stirred for 30 min. 1.35 g
(7.5 mmol) of L-Phe-OMe was added and the reaction mixture
was allowed to attain room temperature slowly. The mixture was
further stirred for 10 h. The precipitate was filtered and the
filtrate was washed three times each with 1 M KHSO4 solution,
sodium carbonate solution and brine. The organic layer was
dried over anhydrous sodium sulfate and solvent was removed
under reduced pressure. The compound was purified by flash
chromatography (hexane : ethyl acetate; 2 : 1). Yield 1.48 g
1
(74%); H NMR (CDCl3) δ (ppm): 1.44 (s, 9H), 1.79–1.95 (m,
3H), 2.98–3.04 (m, 2H), 3.17–3.29 (m, 3H), 3.73 (s, 3H),
4.27–4.30 (dd, 1H), 4.87–4.90 (dd, 1H), 7.09–7.11 (d, 2H),
7.26–7.29 (m, 3H); 13C NMR (CDCl3) δ (ppm): 24.9, 28.7,
31.1, 38.5, 47.4, 52.7, 61.4, 81.2, 127.6, 129.0, 129.7, 136.4,
155.0, 156.1, 172.1; ESI-MS: m/z calcd for C20H28N2O5
[M + Na]+ 399.1896, found 399.3571.
Conclusions
This study on the inhibition of angiotensin converting enzyme
(ACE) by Sec–Pro–Phe and Cys–Pro–Phe tripeptides indicate
that the presence of a Phe residue at the C-terminal of the Sec–
Pro and Cys–Pro dipeptides enhances the inhibitory potency of
the parent compounds. Replacement of the terminal Phe residue
by aliphatic amino acid residues such as Val or Ala dramatically
decreases the inhibition activity. Although Val and Ala are
hydrophobic in nature, the inefficient inhibition by compounds 9
and 10 indicates that the hydrophobic pocket at the active site of
ACE is specific to aromatic amino acid residues. The presence of
Pro residue is crucial for ACE inhibition as the replacement of
this residue by Val almost completely abolishes the ACE inhi-
bition potency. It was also observed that the ring size of Pro
plays an important role in the enzyme inhibition as the replace-
ment of the five-membered ring in compounds 3 and 4 by a six-
membered ring, (e.g. compounds 12 and 13) reduces the ACE
inhibition activity by more than 20 times. This is probably due
to a different orientation of the C- and N-terminal moieties at the
active site. The inhibition of PN-mediated nitration reaction and
GPx-like antioxidant activity of these peptides indicate that the
selenocysteine-containing peptides may provide better protection
against oxidative damage as compared to the cysteine-containing
peptides. In contrast to ACE inhibition, the presence of other
amino acid residues attached to Cys or Sec does not have any
noticeable effect on their antioxidant behavior.
Synthesis of 1523
Compound 15 was synthesized following a similar method given
1
for compound 14 using L-Val–OMe in place of L-Phe–OMe. H
NMR (CDCl3) δ (ppm): 0.74–0.78 (dd, 6H), 1.33 (s, 9H),
1.74–1.78 (m, 3H), 2.01–2.04 (m, 2H), 3.21–3.29 (m, 2H), 3.58
(s, 3H), 4.14–4.17 (dd, 1H), 4.33–4.35 (dd, 1H); 13C NMR
(CDCl3) δ (ppm): 17.5, 19.0, 24.6, 28.3, 31.0, 47.0, 52.0, 57.2,
59.6, 61.0, 80.6, 154.6, 155.8, 172.1; ESI-MS: m/z calcd for
C16H28N2O5 [M + Na]+ 351.1896, found 351.5418.
Synthesis of 1624
Compound 16 was synthesized following a similar method given
1
for compound 14 using L-Ala–OMe in place of L-Phe–OMe. H
NMR (CDCl3) δ (ppm): 1.39–1.40, (d, 3H), 1.47 (s, 9H),
1.69–1.75 (m, 3H), 2.14–2.18 (m, 1H), 3.45–3.49 (m, 2H), 3.75
(s, 3H), 4.18–4.21 (dd, 1H), 4.54–4.57 (dd, 1H); 13C NMR
(CDCl3) δ (ppm): 17.5, 19.0, 24.6, 28.3, 31.0, 47.0, 52.0, 57.2,
61.0, 80.6, 154.6, 155.8, 172.1; ESI-MS: m/z calcd for
C14H24N2O5 [M + Na]+ 323.1523, found 322.9257.
2242 | Org. Biomol. Chem., 2012, 10, 2237–2247
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Synthesis of 1722
55.7, 60.7, 80.6, 114.5, 127.5, 128.1, 129.0, 130.7, 131.1, 136.6,
155.5, 159.1, 171.0, 171.7, 172.2; 77Se NMR (CDCl3) δ (ppm):
222; ESI-MS: m/z calcd for C31H41N3O7Se [M + Na]+
670.2007, found 670.2640.
To 1.40 g (3.72 mmol) of compound 14 was dissolved in 5 mL
chloroform and 14 mL of formic acid was added. The mixture
was stirred for 3 h. Excess solvent was evaporated under reduced
pressure. Yield 0.84 g (82%); 1H NMR (D2O) δ (ppm):
1.77–1.98 (m, 3H), 2.15–2.21 (m, 1H), 2.80–2.86 (m, 1H),
3.03–3.18 (m, 3H), 3.59 (s, 3H), 4.04–4.05 (dd, 1H), 4.20–4.22
(dd, 1H); 13C NMR (CDCl3) δ (ppm): 24.6, 30.5, 37.6, 47.5,
53.1, 55.0, 60.1, 127.8, 129.1, 129.5, 135.9, 169.1, 172.0;
ESI-MS: m/z calcd for C15H21N2O3 [M + H]+ 277.1552, found
276.9391.
Synthesis of 23
Compound 23 was synthesized by following a similar method
given for the synthesis of compound 22 by using compound 21
in place of compound 20. 1H NMR (CDCl3) δ (ppm): 1.44 (s, 9
H), 1.62–1.65 (m, 1H), 1.73–1.76 (m, 1H), 1.87–1.91 (m, 2H),
2.17–2.20 (m, 2H), 2.51–2.56 (dd, 1H), 2.65–2.70 (dd, 1H),
2.95–2.98 (dd, 1H), 3.11–3.14 (dd, 1H), 3.69 (s, 3H), 3.78 (s,
3H), 3.79 (s, 2H), 4.56–4.58 (m, 2H), 4.79–4.82 (dd, 1H),
6.83–6.85 (d, 2H), 7.09–7.11 (d, 2H), 7.21–7.26 (m, 5H); 13C
NMR (CDCl3) δ (ppm): 18.2, 19.6, 25.8, 28.1, 28.8, 31.1, 34.4,
38.3, 52.8, 53.6, 55.7, 59.1, 81.0, 114.6, 127.7, 129.1, 129.7,
130.5, 131.3, 136.2, 155.9, 159.0, 170.7, 171.4, 172.1; ESI-MS:
m/z calcd for C31H41N3O7S [M + Na]+ 622.2563, found
622.3636.
Synthesis of 1823
Compound 18 was synthesized by following a similar method
given for compound 17 but using compound 15. 1H NMR
(CDCl3) δ (ppm): 0.94–0.96 (dd, 6H), 1.96–2.10 (m, 3H),
2.18–2.22 (m, 1H), 2.44–2.47 (m, 1H), 3.40–3.43 (t, 2H), 3.74
(s, 3H), 4.42–4.45 (dd, 1H), 4.77–4.81 (dd, 1H); 13C NMR
(CDCl3) δ (ppm): 17.5, 19.0, 24.6, 28.3, 31.0, 47.0, 52.0, 57.2,
59.6, 61.0, 154.6, 155.8; ESI-MS: m/z calcd for C11H20N2O3
[M + Na]+ 251.1372, found 251.0129.
Synthesis of 24
This compound was synthesized by following a procedure
Synthesis of 1924
similar to the synthesis given for compound 22 but using com-
1
pound 18 in place of compound 17. H NMR (CDCl3) δ (ppm):
This compound was synthesized by following a similar method
given for the synthesis of 17 but using compound 16. H NMR
0.83–0.91 (dd, 6H), 1.43 (s, 9H), 1.90–1.93 (m, 2H), 2.09–2.14
(m, 1H), 2.34–2.37 (m, 1H), 2.60–2.66 (m, 1H), 2.76–2.80 (m,
1H), 3.47–0.349 (m, 1H), 3.58–3.60 (dd, 1H), 3.70 (s, 3H), 3.77
(s, 3H), 3.79 (s, 2H), 4.40–4.44 (m, 2H), 4.59–4.61 (d, 1H),
6.80–6.82 (d, 2H), 7.22–7.24 (d, 2H); 13C NMR (CDCl3) δ
(ppm): 18.4, 19.6, 25.4, 26.1, 27.9, 28.0, 28.8, 31.5, 48.1, 52.1,
52.6, 55.7, 57.9, 60.6, 80.5, 114.4, 130.6, 131.0, 155.6, 159.0,
171.1, 172.0, 172.6; 77Se NMR (CDCl3) δ (ppm): 224; ESI-MS:
m/z calcd for C27H41N3O7Se [M + Na]+ 622.2007, found
622.1271.
1
(CDCl3) δ (ppm): 1.43–1.45 (d, 3H), 2.00–2.16 (m, 3H),
2.44–2.49 (m, 1H), 3.41–3.45 (t, 2H), 3.75 (s, 3H), 4.50–4.54
(dd, 1H), 4.72–4.74 (dd, 1H); 13C NMR (CDCl3) δ (ppm): 16.4,
24.1, 27.9, 30.0, 48.9, 52.4, 59.8, 168.6, 172.8; ESI-MS: m/z
calcd for C9H17N2O3 [M + H]+ 20.11239, found 200.9671.
Synthesis of 20 and 21
Compounds 20 and 21 were synthesized following the literature
procedures.6
Synthesis of 25
This compound was synthesized by following a procedure
Synthesis of 22
similar to the synthesis of compound 22 but using compound 19
1
in place of 17. H NMR (CDCl3) δ (ppm): 1.23–1.25 (d, 3H),
To a 100 mL solution of chloroform, 0.58 g (1.5 mmol) of 20,
0.46 g (2.25 mmol) DCC and 0.34 g (2.25 mmol) HOBt were
added and the mixture was stirred for 20 min at 0 °C. After this,
0.83 g (3 mmol) 17 was added and the reaction mixture was
allowed to attain room temperature slowly. The mixture was
further stirred for 10 h. The precipitate was filtered and the
filtrate was washed three times each with 1 M KHSO4 solution,
sodium carbonate solution and brine. The organic layer was
dried over anhydrous sodium sulfate and solvent was removed
under reduced pressure. The compound was purified by flash
chromatography (hexane : ethyl acetate; 1 : 2). Yield 0.59 g
1.43 (s, 9H), 1.91–1.96 (m, 3H), 2.38–2.41 (m, 1H), 2.67–2.68
(m, 1H), 2.81–2.83 (m, 1H), 3.47–3.49 (m 1H), 3.57–3.59 (m,
1H), 3.71 (s, 3H), 3.78 (s, 3H), 3.79 (s, 2H), 4.46–4.48 (dd, 1H),
4.59–4.63 (m, 2H), 6.80–6.82 (d, 2H), 7.22–7.24 (d, 2H); 13C
NMR (CDCl3) δ (ppm): 18.6, 25.3, 26.0, 27.6, 28.8, 48.0, 48.6,
52.1, 52.9, 55.8, 60.5, 80.6, 114.5, 130.6, 131.1, 155.5, 159.0,
170.8, 171.8, 173.6; 77Se NMR (CDCl3) δ (ppm): 220; ESI-MS:
m/z calcd for C25H37N3O7Se [M + Na]+ 594.1694, found
594.2136.
1
(61%). H NMR (CDCl3) δ (ppm): 1.44 (s, 9 H), 1.60–1.77 (m,
Synthesis of 26
5H), 2.14–2.18 (m, 1H), 2.56–2.59 (dd, 1H), 2.68–2.70 (dd,
1H), 3.01–3.04 (dd, 1H), 3.12–3.15 (dd, 1H), 3.69 (s, 3H), 3.77
(s, 3H), 3.79 (s, 2H), 4.55–4.57 (m, 2H), 4.79–4.81 (dd, 1H),
6.81–6.83 (d, 2H), 7.06–7.27 (m, 7H); 13C NMR (CDCl3) δ
(ppm): 25.2, 27.0, 28.8, 30.2, 33.7, 38.4, 47.9, 51.9, 52.2, 53.8,
To a 15 mL methanolic solution of 22 (0.32 g, 0.5 mmol),
iodine (0.19 g, 0.75 mmol) was added. Water (15 mL) was
added to the reaction mixture and stirred for 15 min. To this,
0.5 mL of hydrazine hydrate was added to decompose the excess
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1
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 0.22 g
(86%);1H NMR (CDCl3) δ (ppm): 1.23–1.25 (m, 4H), 1.41 (s,
9H), 1.87–1.92 (m, 2H), 2.16–2.19 (m, 1H), 3.02–3.04 (dd, 1H),
3.10–3.13 (m, 2H), 3.16–3.20 (m, 1H), 3.56–3.59 (m, 1H), 3.69
(s, 3H), 4.53–4.54 (d, 1H), 4.70–4.80 (m, 2H), 7.11–7.13 (d,
2H), 7.19–7.24 (m, 3H); 13C NMR (CDCl3) δ (ppm): 14.6,
25.2, 28.8, 30.1, 32.9, 38.3, 48.0, 52.8, 53.8, 60.7, 80.7, 127.6,
129.0, 129.7, 136.4, 157.6, 171.1, 171.3, 172.3; 77Se NMR
(CDCl3) δ (ppm): 301; ESI-MS: m/z calcd for C46H64N6O12Se2
[M + Na]+ 1073.2818, found 1073.2656.
Yield 0.14 g (86%); H NMR (MeOH-d4) δ (ppm): 0.85–0.89
(m, 4H), 1.85–1.90 (m, 2H), 2.12–2.15 (m, 1H), 2.99–3.01 (dd,
1H), 3.10–3.14 (m, 2H), 3.11–3.13 (m, 1H), 3.56–3.59 (m, 1H),
3.70 (s, 1H), 4.44–4.46 (d, 1H), 4.70–4.75 (m, 2H), 7.12–7.14
(d, 2H), 7.19–7.25 (m, 3H); 13C NMR (MeOH-d4) δ (ppm):
24.9, 27.7, 29.4, 29.7, 37.3, 51.7, 54.4, 60.5, 78.4, 126.9, 128.5,
129.4, 136.8, 166.4, 172.2, 172.6; 77Se NMR (MeOH-d4) δ
(ppm): 292; ESI-MS: m/z calcd for C36H48N6O8Se2 [M + Na]+
875.1762, found 875.1169.
Synthesis of 31
The synthetic procedure followed for the synthesis of compound
31 is similar to the method given for diselenide 30. H NMR
1
(MeOH-d4) δ (ppm): 1.74–1.82 (m, 4H), 2.58–2.64 (m, 1H),
2.91–2.98 (m, 3H), 3.20–3.26 (m, 2H), 3.55 (s, 3H), 4.08–4.09
(d, 1H), 4.51–4.54 (dd, 1H), 4.86–4.91 (dd, 1H), 6.78–6.80 (d,
2H), 7.08–7.25 (m, 3H); 13C NMR (MeOH-d4) δ (ppm): 24.7,
29.3, 31.1, 35.2, 37.3, 51.1, 51.6, 54.7, 60.6, 114.1, 128.5,
129.3, 136.8, 159.6, 166.8, 172.1; ESI-MS: m/z calcd for
C36H48N6O8S2 [M + H]+ 757.3053, found 757.2333.
Synthesis of 27
Compound 27 was synthesized by following a similar method
given for compound 26. H NMR (CDCl3) δ (ppm): 1.47 (s,
1
9H), 1.55–1.58 (m, 1H), 1.78–1.81 (m, 1H), 1.92–1.94 (m, 2H),
2.55–2.60 (m, 2H), 2.69–2.71 (dd, 1H), 3.00–3.02 (dd, 1H),
3.14–3.16 (dd, 1H), 3.45–3.49 (m, 1H), 3.72 (s, 3H), 4.60–4.63
(m, 2H), 4.83–4.85 (dd, 1H), 7.12–7.15 (m, 2H), 7.24–7.32 (m,
3H); 13C NMR (CDCl3) δ (ppm): 25.0, 28.8, 34.4, 36.4, 38.3,
48.0, 52.8, 53.7, 55.8, 60.7, 80.6, 128.9, 129.5, 130.6, 136.7,
159.3, 171.0, 171.6, 172.2; ESI-MS: m/z calcd for
C46H64N6O12S2 [M + Na]+ 979.3921, found 980.3397.
Synthesis of 32
This compound was synthesized by following a similar pro-
1
cedure given for compound 30 but using the tripeptide 28. H
NMR (MeOH-d4) δ (ppm): 0.83–0.85 (dd, 6H), 1.15–1.18 (m,
2H), 1.89–2.01 (m, 5H), 3.35–3.28 (m, 1H), 3.44–3.48 (m, 1H),
3.58 (s, 3H), 4.18–4.20 (dd, 1H), 4.41–4.48 (m, 2H); 13C NMR
(MeOH-d4) δ (ppm): 17.7, 18.6, 25.1, 29.8, 31.6, 37.4, 40.5,
51.7, 54.6, 58.9, 78.5, 167.8, 172.2, 172.4; 77Se NMR (MeOH-
d4) δ (ppm): 292; ESI-MS: m/z calcd for C28H48N6O8Se2
[M + H]+ 757.1942, found 757.2333.
Synthesis of 28
This compound was synthesized by following a similar pro-
cedure given for compound 26 but using the tripeptide 24. H
1
NMR (CDCl3) δ (ppm): 0.88–0.90 (dd, 6H), 1.23–1.25 (m, 2H),
1.41 (s, 9H), 1.97–2.13 (m, 4H), 2.27–2.29 (m, 1H), 3.20–3.27
(m, 2H), 3.69 (s, 3H), 4.42–4.46 (dd, 1H), 4.59–4.61 (dd, 1H),
4.75–4.79 (m, dd, 1H); 13C NMR (CDCl3) δ (ppm): 18.4, 19.6,
25.5, 28.8, 30.6, 31.5, 32.5, 48.2, 52.6, 52.9, 57.8, 60.8, 80.7,
155.7, 171.1, 171.4, 172.7; 77Se NMR (CDCl3) δ (ppm): 299;
ESI-MS: m/z calcd for C38H64N6O12Se2 [M + Na]+ 979.2810,
found 979.2982.
Synthesis of 33
This compound was synthesized by following a similar pro-
1
cedure given for compound 30 but using the tripeptide 29. H
NMR (MeOH-d4) δ (ppm): 1.21–1.22 (d, 3H), 1.96–2.03 (m,
3H), 2.23–2.27 (m, 2H), 3.25–3.26 (m, 2H), 3.65 (s, 3H),
4.31–4.34 (dd, 1H), 4.43–4.49 (m, 2H); 13C NMR (MeOH-d4) δ
(ppm): 16.2, 24.9, 27.4, 29.5, 29.8, 51.8, 52.4, 60.5, 78.4,
166.4, 172.6, 173.6; 77Se NMR (MeOH-d4) δ (ppm): 292;
ESI-MS: m/z calcd for C24H40N6O8Se2 [M + H]+ 701.1316,
found 701.1487.
Synthesis of 29
This compound was synthesized by following a similar pro-
cedure given for compound 26 but using the tripeptide 25. H
1
NMR (CDCl3) δ (ppm): 1.21–1.22 (d, 3H), 1.39 (s, 9H),
1.97–2.02 (m, 3H), 2.05–2.08 (m, 2H), 3.16–3.19 (m, 1H),
3.26–3.29 (m, 1H), 3.69 (s, 3H), 4.45–4.54 (m, 2H), 4.74–4.76
(dd, 1H); 13C NMR (CDCl3) δ (ppm): 18.6, 25.4, 28.8, 30.1,
32.1, 32.8, 48.1, 48.6, 52.9, 60.6, 80.6, 155.7, 171.0, 173.7;
77Se NMR (CDCl3) δ (ppm): 300; ESI-MS: m/z calcd for
C34H56N6O12Se2 [M + Na]+ 923.2184, found 923.1868.
Synthesis of 3425
To a solution of Boc-L-Val (1.8 g, 8.25 mmol) in chloroform
(130 mL), DCC (2.52 g, 12.38 mmol) followed by HOBt
(1.89 g, 12.38 mmol) was added at 0 °C and the reaction
mixture was stirred for 20 min. To this L-Phe–OMe (2.22 g,
12.38 mmol) was added followed by triethylamine (2.3 mL,
16.5 mmol). The reaction mixture was slowly allowed to attain
room temperature and further stirred for 12 h. After the com-
pletion of the reaction, the precipitate was filtered and the chloro-
form layer was washed two times each with 1 M aqueous
KHSO4 solution, 1 M aqueous Na2CO3 solution and brine
Synthesis of 30
Compound 26 (0.20 g, 0.19 mmol) was taken in a round-bot-
tomed flask and 1 mL of trifluoroacetic acid (TFA) was added.
The flask was sealed and stirred for 3 h. TFA was evaporated
under reduced pressure to obtain the Boc free dipeptide 30.
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solution successively. Solvent was evaporated under reduced
pressure and the compound was purified with column chromato-
graphy (pet. ether : ethyl acetate; 3 : 1). Yield:1.66 g (53.1%); 1H
NMR (CDCl3) δ: 0.86–0.93 (m, 6H), 1.45 (s, 9H), 2.05–2.13
(m, 1H), 3.11–3.14 (t, 2H), 3.72 (s, 3H), 3.88–3.92 (t, 1H),
4.85–4.90 (q, 1H), 7.10–7.12 (t, 2H), 7.24–7.31 (m, 3H); 13C
NMR (CDCl3) δ: 18.2, 19.6, 28.8, 31.4, 38.4, 52.8, 53.6, 60.3,
80.4, 127.7, 129.1, 129.7, 136.1, 171.8, 172.2; ESI-MS: m/z
calcd for C20H30N2O5 [M + Na]+ 401.4524, found 400.9930.
127.0, 128.6, 129.2, 136.9, 167.7, 172.1, 172.4; ESI-MS m/z
calcd for C36H52N6O8S2 [M + H]+ 761.3366, found 761.3551.
Synthesis of 39
To a solution of 20 (0.79 g, 2.0 mmol) in 120 mL chloroform,
DCC (0.62 g, 3 mmol) and HOBt (0.46 g, 3.0 mmol) were
added at 0 °C and stirred for 20 min. S-Methyl-piperidine-2-car-
boxylate (0.43 g, 3.0 mmol) was added to this and the mixture
was stirred at room temperature for 10 h. The precipitate was
filtered and the filtrate was washed three times successively with
1 M KHSO4 solution, 1 M Na2CO3 solution and brine. The
solvent was evaporated and the compound was purified by
column chromatography (hexane : ethyl acetate; 1 : 2). Yield
0.62 g (59%); 1H NMR (CDCl3) δ (ppm): 1.44 (s, 9H),
1.57–1.63 (m, 6H), 2.23–2.26 (d, 1H), 2.59–2.64 (m, 1H),
2.79–2.84 (m, 1H), 3.03–3.06 (t, 1H), 3.69 (s, 3H), 3.76–3.79
(m, 5H), 4.83–4.84 (dd, 1H), 5.33–5.34 (d, 1H), 6.79–6.81 (d,
2H), 7.22–7.24 (d, 2H); 13C NMR (CDCl3) δ (ppm): 21.4, 25.0,
25.6, 26.9, 27.7, 28.8, 40.6, 44.0, 50.6, 52.8, 55.7, 80.2, 114.3,
130.6, 131.4, 155.6, 158.8, 171.3, 171.7; 77Se NMR (CDCl3) δ
(ppm) 222; ESI-MS: m/z calcd for C23H34N2O6Se [M + Na]+
537.1480, found 537.1052.
Synthesis of 3525
To a solution of 34 (1.66 g, 4.4 mmol) in 5 mL chloroform,
trifluoroacetic acid (4.4 mL) was added. The reaction mixture was
stirred for 4 h. Solvent was evaporated under reduced pressure.
1
Yield:1.03 g (84%); H NMR (CDCl3) δ (ppm): 0.87–1.0 (m,
6H), 2.14 (m, 1H), 3.02–3.08 (m, 2H), 3.67 (s, 3H), 3.93 (m,
1H), 4.69–4.71 (d, 1H), 7.09–7.10 (d, 2H), 7.20–7.31 (m, 3H);
13C NMR (CDCl3) δ (ppm): 18.0, 18.1, 30.8, 37.5, 53.0, 54.8,
59.3, 127.9, 129.2, 129.5, 135.6, 171.9; ESI-MS m/z calcd for
C15H22N2O3 [M + H]+ 279.1709, found 278.9557.
Synthesis of 36
This compound was synthesized by following a route similar to
the synthesis of 22 but using compounds 35 and 21 as starting
1
material. H NMR (CDCl3) δ (ppm): 0.83–0.91 (dd, 6H), 1.44
Synthesis of 40
(s, 9H), 2.73–2.75 (dd, 1H), 2.84–2.86 (dd, 1H), 3.08–3.12 (m,
2H), 3.70 (s, 2H), 3.71 (s, 3H), 3.78 (s, 3H), 4.19–4.22 (m, 2H),
4.85–4.87 (dd, 1H), 6.84–6.86 (d, 2H), 7.08–7.10 (d, 2H),
7.22–7.27 (m, 5H); 13C NMR (CDCl3) δ (ppm): 18.1, 19.6,
25.4, 28.8, 31.0, 34.4, 36.5, 38.3, 52.8, 53.6, 55.7, 59.1, 81.0,
114.6, 127.7, 129.1, 129.7, 130.3, 136.1, 155.9, 157.2, 159.3,
170.7, 171.1, 172.1; ESI-MS m/z calcd for C31H43N3O7S
[M + Na]+ 624.2719, found 624.4609.
To a solution of 21 (0.68 g, 2.0 mmol) in 120 mL chloroform,
DCC (0.62 g, 3.0 mmol) and HOBt (0.46 g, 3.0 mmol) were
added at 0 °C and stirred for 20 min. S-Methyl-piperidine-2-car-
boxylate (0.43 g, 3.0 mmol) was added to this and the mixture
was stirred at room temperature for 10 h. The precipitate was
filtered and the filtrate was washed three times each with 1 M
KHSO4 solution, 1 M Na2CO3 solution and brine. The solvent
was evaporated and the compound was purified by column
chromatography (hexane : ethyl acetate; 1 : 2). Yield 0.69 g
Synthesis of 37
1
(74%). H NMR (CDCl3) δ (ppm): 1.46 (s, 9H), 1.57–1.63 (m,
4H), 1.71–1.75 (m, 2H), 2.24–2.28 (m, 1H), 2.49–2.54 (m, 1H),
2.77–2.82 (m, 1H), 3.03–3.06 (t, 1H), 3.70 (s, 3H), 3.79–3.81
(m, 5H), 4.82–4.84 (dd, 1H), 5.34–5.35 (d, 1H), 6.83–6.85 (d,
2H), 7.26–7.28 (d, 2H); 13C NMR (CDCl3) δ (ppm): 21.3, 25.6,
26.9, 27.7, 28.8, 34.0, 36.2, 43.9, 50.2, 52.8, 55.7, 80.2, 114.3,
130.3, 130.6, 155.8, 159.1, 171.3, 171.7; ESI-MS: m/z calcd for
C23H34N2O6S [M + Na]+ 489.2035, found 489.1072.
This disulfide was synthesized by following a method similar to
the synthesis of the diselenide 26 but using the tripeptide 36. 1H
NMR (CDCl3) δ (ppm): 0.96–0.98 (dd, 6H), 1.48 (s, 9H),
1.70–1.75 (m, 1H), 1.93–1.97 (m, 1H), 2.11–2.14 (m, 1H),
3.07–3.10 (m, 2H), 3.71 (s, 3H), 4.36–4.38 (dd, 1H), 4.64–4.66
(dd, 1H), 4.88–4.90 (dd, 1H), 7.14–7.16 (d, 2H), 7.24–7.31 (m,
3H); 13C NMR (CDCl3) δ (ppm): 19.1, 19.6, 25.4, 26.0, 28.8,
34.3, 38.3, 52.7, 53.8, 59.6, 80.6, 127.6, 129.1, 129.7, 136.3,
155.8, 171.2, 171.6, 172.3; ESI-MS m/z calcd for
C46H68N6O12S2 [M + Na]+ 983.4234, found 983.3778.
Synthesis of 41
This compound was synthesized following a similar method
Synthesis of 38
given for compound 26 using compound 39 as the starting
1
This compound was synthesized by following a similar pro-
cedure given for compound 30 but using the tripeptide 37. H
material. H NMR (CDCl3) δ (ppm): 1.44 (s, 9H), 1.68–1.76
1
(m, 5H), 2.27–2.30 (d, 1H), 3.21–3.31 (m, 3H), 3.73 (s, 3H),
3.96–3.99 (m, 1H), 4.98–5.01 (dd, 1H), 5.35–5.37 (d, 1H); 13C
NMR (CDCl3) δ (ppm): 21.3, 25.5, 26.8, 28.7, 33.1, 40.5, 44.0,
51.0, 52.7, 80.3, 155.5, 170.9, 171.6; 77Se NMR (CDCl3) δ
(ppm) 309; ESI-MS: m/z calcd for C30H50N4O10Se2 [M + Na]+
809.1755, found 809.1743.
NMR (MeOH-d4) δ (ppm): 0.83–0.85 (dd, 6H), 1.71–1.73 (m,
1H), 1.93–1.96 (m, 1H), 2.81–2.84 (m, 1H), 2.96–2.99 (m, 1H),
3.15–3.17 (d, 1H), 3.51 (s, 3H), 4.25 (dd, 1H), 4.38–4.40 (dd,
1H), 4.52–4.54 (dd, 1H), 7.03–7.11 (m, 5H); 13C NMR (MeOH-
d4) δ (ppm): 17.7, 18.6, 25.0, 29.7, 31.5, 37.3, 54.5, 58.8, 78.4,
This journal is © The Royal Society of Chemistry 2012
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Synthesis of 42
Nitration of angiotensin II
Compound 42 was synthesized by following a procedure similar
to the synthesis of compound 26 by using compound 40. H
High performance liquid chromatography (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 μm)
with isocratic ellution 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 μM) and peroxynitrite (300 μM) in
sodium phosphate buffer (100 mM) of pH 7.5 without and with
increasing concentration of the inhibitor was 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.
1
NMR (CDCl3) δ (ppm): 1.36 (s, 9H), 1.60–1.68 (m, 5H),
2.19–2.22 (m, 2H), 3.01–3.06 (m, 1H), 3.15–3.20 (t, 1H), 3.65
(s, 3H), 3.91–3.94 (m, 1H), 4.82–4.84 (dd, 1H), 5.28–5.29 (dd,
1H); 13C NMR (CDCl3) δ (ppm): 21.3, 25.5, 26.7, 28.6, 41.7,
43.9, 52.6, 56.6, 80.2, 155.5, 170.8, 171.6; ESI-MS: m/z calcd
for C30H50N4O10S2 [M + Na]+ 713.2866, found 713.2884.
Synthesis of 43
Compound 43 was synthesized following a procedure similar to
the synthesis of 30 but using compound 41 as starting material.
1H NMR (CDCl3) δ (ppm): 1.30–1.33 (m, 1H), 1.52–1.73 (m,
3H), 2.25–2.28 (m, 1H), 3.21–3.31 (m, 2H), 3.44–3.48 (m, 2H),
3.71 (s, 3H), 3.82–3.86 (m, 1H), 4.82–4.84 (dd, 1H), 5.27–5.29
(d, 1H); 13C NMR (MeOH-d4) δ (ppm): 20.8, 25.1, 26.4, 34.1,
43.8, 51.1, 52.2, 53.1, 167.7, 171.5; 77Se NMR (MeOH-d4) δ
(ppm) 286; ESI-MS: m/z calcd for C20H34N4O6Se2 [M + Na]+
609.0706, found 609.1763.
Measurement of GPx activity
Synthesis of 44
GPx activity of the test compounds was carried out by following
a literature procedure with minor modifications.16b In brief, the
reaction mixture contained glutathione (4.0 mM, glutathione
disulfide reductase (GR, 1.7 units mL−1), and nicotinamide
adenine dinucleotide phosphate (NADPH; 0.4 mM) in 100 mM
potassium phosphate buffer of pH 7.5. The samples (100 μM)
were added to the reaction mixture at 22 °C, and the reaction
was started by addition of H2O2 (5 mM). The initial reduction
rates (v0) were calculated from the rate of NADPH oxidation at
340 nm. Each initial rate was measured at least 3 times and cal-
culated from the first 5–10% of the reaction by using the molar
extinction coefficient (6.22 mM−1 cm−1) for NADPH. The
initial rates were corrected for a background reaction between
peroxide and glutathione.
This compound was synthesized following a similar method as
reported for compound 30 but using compound 42. H NMR
1
(MeOH-d4) δ (ppm): 1.32–1.35 (m, 1H), 1.69–1.76 (m, 3H),
2.27–2.30 (m, 1H), 3.01–3.07 (m, 1H), 3.28–3.38 (m, 3H), 3.72
(s, 3H), 3.87–3.90 (m, 1H), 4.91–4.94 (dd, 1H), 5.29–5.30 (d,
1H); 13C NMR (MeOH-d4) δ (ppm): 20.8, 25.1, 26.4, 26.8,
36.3, 43.8, 52.2, 53.2, 167.6, 171.5; ESI-MS: m/z calcd for
C20H34N4O6S2 [M + Na]+ 513.1817, found 513.3591.
Synthesis of peroxynitrite (PN)
Peroxynitrite was synthesized by following the literature method
with minor modifications.6
Acknowledgements
ACE assay
This study was supported by the Department of Science and
Technology (DST), New Delhi. G. M. acknowledges the DST
for the award of Ramanna and Swarnajayanti fellowships and
B. J. B. thanks the Council of Scientific and Industrial Research,
New Delhi and Indian Institute of Science, Bangalore for a
research fellowship.
The assay was performed in 400 μL sample vials and an auto-
sampler was used for sample injection. Ang I and AngII were
analyzed by reverse-phase HPLC (Princeton C18 column, 4.6 ×
150 mm, 5 μm) with isocratic elution of 50 : 50 MeOH : 0.1%
TFA in water. In the ACE inhibition assay, we employed a
mixture of 50 μM 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 incu-
bated 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. The 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.
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