Solid-phase total synthesis of cyclic peptide brachystemin A and its immunomodulating activity

Article abstract of DOI:10.3906/kim-1412-58

Brachystemin A (1) is a biologically active peptide from a Chinese traditional plant Brachystemma calycinum D. Don. (Caryophyllaceae). The current study describes the complete solid-phase total synthesis of peptide 1 by using Kenner's sulfonamide safety-c

Full text of DOI:10.3906/kim-1412-58

Turk J Chem
Turkish Journal of Chemistry
(2015) 39: 930 – 938
¨
http://journals.tubitak.gov.tr/chem/
˙
c
⃝ TUBITAK
doi:10.3906/kim-1412-58
Research Article
Solid-phase total synthesis of cyclic peptide brachystemin A and its
immunomodulating activity
Zafar Ali SHAH1, Almas JABEEN2, Samreen SOOMRO2, M. Ahmed MESAIK3,
M. Iqbal CHOUDHARY1,2,4, Farzana SHAHEEN1,∗
1H.E.J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences,
University of Karachi, Karachi, Pakistan
2Dr. Panjwani Center for Molecular Medicine and Drug Research, University of Karachi, Karachi, Pakistan
3Faculty of Pharmacy, Universiti Kebangsaan Malaysia, Jalan Raja Muda Abdul Aziz, Kuala Lumpur, Malaysia
4Department of Biochemistry, Faculty of Science, King Abdulaziz University, Jeddah, Saudi Arabia
Received: 23.12.2014
•
Accepted/Published Online: 25.03.2015
•
Printed: 30.10.2015
Abstract: Brachystemin A (1) is a biologically active peptide from a Chinese traditional plant Brachystemma calycinum
D. Don. (Caryophyllaceae). The current study describes the complete solid-phase total synthesis of peptide 1 by using
Kenner’s sulfonamide safety-catch linker strategy. It was identified by QTOF/MS data and NMR studies. Synthetic
peptide 1 was tested for its immunomodulatory effect on different inflammatory parameters, including production
of inflammatory cytokines, interleukin 2 (IL-2), tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β), and
generation of nitric oxide (NO.). The synthetic peptide 1 showed a moderate to low level of inhibition on the production
of IL-2 (35.2%), TNF-α (19.3%), and IL-1β (7.5%) at a concentration of 25 µM. The effect of the compound on viability
of cells was also evaluated, and it was found to be nontoxic on 3T3 cells.
Key words: Brachystemma calycinum, cyclic peptide, brachystemin A, safety-catch linker, immunomodulatory effect,
cytokines
1. Introduction
Plants belonging to the family Caryophyllaceae produce cyclic peptides containing 5–9 proteinogenic amino
acids.1−3 Most of them are biologically active in mammalian systems. The medicinal plant Brachystemma
calycinum D.Don. (Caryophyllaceae) is known to contain the cyclopeptides brachystemins A–I. B. calycinum
is used in Chinese folk medicine for the treatment of rheumatoid arthritis, impotence, limb numbness, and
gonorrhea. On the basis of the traditional uses of this herb in inflammatory diseases, the cyclic peptide
constituents of this plant were tested in vitro for their inhibitory effects on the secretion of chemokine ligand-2,
interleukin 6, and collagen IV by high-glucose-stimulated mesangial cells. Brachystemin A (1) was identified as
the most active constituent as it significantly inhibited the secretion of interleukin 6, chemokine ligand-2, and
collagen IV and exhibited no toxic effect in mesangial cells.4,5
Inflammation and oxidative stress are involved in many pathological conditions. The role of inflammatory
mediators, including cytokines and free radicals, in the disease pathology is well established. Various diseases
caused by deregulation of the immune system, including rheumatoid arthritis, atherosclerosis, inflammatory
∗Correspondence: afnan.iccs@gmail.com
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SHAH et al./Turk J Chem
bowel disease, diabetes, and neurodegenerative disorders, pose serious health problems worldwide. New thera-
peutic approaches to target inflammatory parameters, mainly involved in disease pathogenesis, are continuously
needed.6
Biologically active cyclic peptides are reported from many natural sources.7,8 However, they are usually
obtained from natural sources in very low yields. Many investigators have reported the synthesis of cyclic
peptides by different strategies9−12 to obtain them in sufficient quantities for subsequent development as
medicinal agents. The yield of cyclic peptides through various cyclization approaches are often low due to many
reasons, including difficulties in the cyclization of peptides having three to eight amino acids on resin,13−17
epimerization at the C -terminal amino acid, and the formation of cyclic dimers and oligomers.18,19 Among the
various synthetic methods, the on-resin cyclization approach has been successfully employed in the synthesis
of many biologically active natural products.20−23 The current study describes the solid-phase synthesis of
brachystemin A (1) by using Kenner’s sulfonamide safety-catch linker. The synthetic peptide was evaluated for
its immunomodulatory activity by performing different cell-based assays.
2. Results and discussion
Brachystemin A (1) was first isolated from B. calycinum by Cheng and coworkers. It was identified as cyclo-
Pro1 -Phe2 -Leu3 -Ala4 -Thr5 -Pro6 -Ala7 -Gly8 . Later, this compound was re-isolated and its primary structure
was revised as cyclo-Pro1 -Pro2 -Ala3 -Gly4 -Leu5 -Ala6 -Thr7 -Phe8 by quadrupole-time-of-flight (QTOF) mass
spectrometers and X-ray diffraction analysis. During the course of the current study, Lijing Fang et al. reported
the combination of solid and solution-phase synthesis of brachystemin A in which triphosgene (BTC) was used
as the coupling agent in the synthesis of the linear precursor of brachystemin A. The linear peptide was cleaved
off from the Wang resin and finally subjected to cyclization.
Herein, we report another convenient route to the complete solid-phase synthesis of peptide 1 by using
resin bound sulfonamide anchor (Scheme). The use of sulfonamide linker allows the cyclization and cleavage
steps simultaneously from the resin, thus minimizing the formation of side products. The first amino acid is
usually loaded twice on the solid-supported safety-catch linker to ensure maximum loading. In order to avoid
the expected racemization during the loading step, as well as to increase the yield of target peptide 1, the less
sterically hindered amino acid residue of 1, i.e. glycine, was selected as the first amino acid for coupling with
the resin in the synthesis of peptide 1 (Scheme). It is also expected that the terminal free amino group of linear
peptide precursor would more readily attack a less sterically hindered electrophilic carbonyl of glycine during
the macrocyclization step. The two proline residues present in brachystemin A (1) also serve as turn-inducers.24
It would further make the two ends close to each other, and can support macrocyclization.
On the basis of the above consideration, the first amino acid Fmoc-Gly was loaded on sulfonamide resin
by using the coupling agent (benzotriazol-1-yloxy) tripyrrolidino- phosphoniumhexafluorophosphate (PyBOP)
and base N, N -diisopropylethylamine (DIEA). The loading capacity of the first amino acid was analyzed by
UV spectroscopy.25 The linear peptidyl resin 4 was constructed by using the Fmoc protocol. The Fmoc group
of terminal amino acid of peptidyl resin was replaced by the bulkier trityl group before the activation of the
safety-catch linker by cyanomethylation. The terminal amino acid of the linear peptide was made free by using
5% triflouro acetic acid (TFA) in dichloro- methane. The cyclization and cleavage of peptide from resin were
carried out in the presence of DIEA and tetrahydrofuran. The crude peptide was finally deprotected and then
purified by recycling reversed-phase high performance liquid chromatography (RP-HPLC) by using a reverse-
phase (C18) column to obtain cyclic peptide 1. The structure of synthetic peptide 1 was fully characterized by
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SHAH et al./Turk J Chem
Reagents and reaction conditions: (a) PyBOP, DIEA, Fmoc-Gly-OH/DMF, 0 ◦ C, 12 h, repeated twice; (b) (i) 20%
4-methylpiperidine/DMF, 20 min (ii) Fmoc-Ala-OH/DMF, PyBOP, DIEA, 4 h; (c) (i) 20% 4-methylpiperidine/DMF,
20 min (ii) Fmoc-Pro-OH/DMF, PyBOP, DIEA, 4 h; (d) (i) 20% 4-methylpiperidine/DMF, 20 min (ii) Fmoc-Pro-
OH/DMF, PyBOP, DIEA, 4 h; (e) (i) 20% 4-methylpiperidine/DMF, 20 min (ii) Fmoc-Phe-OH/DMF, PyBOP, DIEA,
4 h; (f) (i) 20% 4-methylpiperidine/DMF, 20 min (ii) Fmoc-Thr (O − tbu)-OH/DMF, PyBOP, DIEA, 4 h; (g) (i) 20%
4-methylpiperidine/DMF, 20 min (ii) Fmoc-Ala-OH/DMF, PyBOP, DIEA, 4 h; (h) (i) 20% 4-methylpiperidine/DMF,
20 min (ii) Fmoc-Leu-OH/DMF, PyBOP, DIEA, 4 h; (i) (i) 20% 4-methylpiperidine/DMF, 20 min (ii) trityl chloride,
DIEA; (j) ICH2 CN, DIEA, NMP, 24 h; (k) (i) 5% TFA/DCM, 0.5 h, (ii) DIEA, 20 h in THF, (iii) TFA/TIS/H2 O
(9.5:0.25:0.25).
Scheme 1. Synthesis of brachystemin A (1).
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SHAH et al./Turk J Chem
1D and 2D-NMR, as well as QTOF data. Thus, the current study describes the complete solid-phase synthesis
of brachystemin A via an on-resin cyclization approach. The overall yield of the finally pure product (7.4%)
is higher than that of the earlier reported synthesis of cyclic peptides by using the safety-catch linker strategy
(Figures S1–S8, supporting information; on the journal’s website).
The molecular formula of cyclic peptide 1 was deduced as C37 H54 N8 O9 by high resolution electron spray
ionization (HRESI) mass spectrometry, which exhibited the molecular ion [M+H]+ at m/z 755.4068 (calcd.
755.4092). NMR data were recorded in d5 -pyridine as it was used for natural brachystemin A previously.
A comparative study of 1 H NMR data of natural and synthetic brachystemin A exhibited close resemblance
(Table 1). 1 H NMR showed six amide protons, resonating at δH 10.56 (1H, b, Ala3 -NHCO), 9.60 (1H, d,
Ala6 -NHCO), 8.96 (1H, t, Gly4 -NHCO), 7.70 (1H, d, Phe8 -NHCO), 7.51 (1H, d, Thr7 -NHCO), and 7.15 (1H,
s, Leu5 -NHCO). These amide proton resonances were the same as those observed with natural brachystemin
A. Furthermore, L-amino acid residues of cyclic peptide 1 showed alpha protons at δH 5.39 (1H, Leu5 -CH),
5.21 (1H, m, Ala3 -CH), 5.05 (1H, m, Phe8 -CH), 5.03 (1H, d (J = 6.0 Hz), Pro1 -CH), 4.79 (1H, d, J = 8.1
Hz, Thr7 - CH), 4.60 (1H, m, Pro2 -CH), and 4.29 (1H, m, Ala6 -CH) (Table 1). The 13 C NMR chemical shift
differences of Pro1 (∆δ Cβ -Cγ = 3.1) and Pro2(∆δ Cβ -Cγ = 3.6) indicated that the amide bonds in the two
Pro residues are trans,26 similar to the natural product (Table 1). The structure of cyclic peptide 1 was further
confirmed by QTOF/MS data, which showed a series of bn (+1) ion peaks at m/z 737, 608, 507, 436, and 266,
corresponding to the successive loss of Phe, Thr, Ala, Leu-Gly, and the terminal tripeptide ion Pro-Pro-Ala
(Table 2).
The effect of synthetic peptide 1 was observed on inflammatory cytokines TNF-α, on IL-1β produced
from THP-1 cells, and on IL-2 produced from Jurkat cells at a concentration of 25 µM. All biological assays
were performed in triplicate. Standard deviation values are presented in Table 3. The peptide moderately
inhibited the production of TNF-α(19.3%) and IL-2 (35.2%), whereas a low level of inhibition was also
observed on IL-1β (7.5%). The peptide was also evaluated for its effect on nitric oxide (NO.) generation
by using lipopolysaccharide activated macrophages from the J774.2 cell line. Similar to the natural product,
the synthetic peptide showed a very weak inhibitory effect (7.5%) at a concentration of 30 µM. The peptide
was further evaluated for cytotoxicity against fibroblast cell line 3T3, where it was found to be nontoxic (Table
3). The data for cytotoxicity are plotted as percent viability explaining the number of viable cells at different
concentrations of peptide 1 and standard drug (Figure). The inactivity on nitric oxide (NO.) and noncytotoxic
effect of peptide 1 was in agreement with the previously described results of isolated peptide.
Figure. Effect of brachystemin A (1) on viability of 3T3 cells. Cyclohexamide was used as standard drug for cytotoxicity.
933

SHAH et al./Turk J Chem
Table 1. 1 H and 13 C spectral data of cyclic peptide 1.
Amino
Acid
Position Natural Peptide 1
Synthetic Peptide 1
δ1 H (J in Hz)
5.05 m
3.45 m
3.34 m
δ1 H (J in Hz)
5.01 m
3.45 m
δ13
55.2
36.1
C
δ13
C
55.67
36.42
Phe
α
β
3.34 m
γ
σ
NH
C=O
α
138.9
130.1 129.0 127.2 7.18–7.51*
7.70, d, (J = 5.4)
138.7
129.9 128.8 127.0
7.18–7.53
7.73, d, (J = 5.6)
169.8
59.8
68.0
21.8
169.6
59.5
66.3
21.8
Thr
4.79, d, (J = 8.0)
5.03 m
1.40, d, (J = 6.4)
7.50, d, (J = 7.6)
4.79, d, (J = 8.15)
5.03 m
1.39, d, (J = 6.3)
7.51, d, (J = 7.6)
β
CH3γ
NH
C=O
α
CH3
NH
C=O
α
172.2
53.6
16.8
171.7
53.4
16.7
Ala6
Leu
4.30 m
1.55, d, (J = 5.6)
10.61, d, (J = 2.0)
4.29 m
1.55, d, (J = 5.2)
10.56, bs
174.2
49.7
29.1
173.7
49.0
28.1
5.43 m
1.31m
1.25m
2.03m
5.39 m
1.31 m
1.24 m
2.03m
β
γ
24.8
24.5
2CH3σ
0.89, d, J (6.8), 0.99, 21.3, 23.8
d, J =(6.0)
7.10( br. s)
0.73, d, J (6.3), 20.9, 24.5
0.97, d, J = (6.3)
7.15 (bs)
NH
C=O
α
177.2
177.17
44.1
Gly
4.54, dd, (J = 16.8, 44.2
5.6) 3.87 dd (16.8, 5.6)
3.87, (J = 16.8,5.6)
8.96, t, (J = 6.4)
169.39
4.53, Overlap
β
3.86, Overlap
8.96, t, (J = 6.0)
NH
C=O
α
CH3
NH
C=O
α
169.3
48.6
18.7
Ala3
Pro2
5.20 (m)
1.84, d, (J = 7.6)
7.95, d, (J = 9.6)
48.8
18.8
5.21(m)
1.85, d, (J = 7.2)
7.95, d, (J = 9.6)
173.7
59.8
30.0
26.4
173.5
63.9
28.9
26.2
4.61 m
2.02 m
1.55 m
1.32 m
3.16 m
3.38 m
4.60 m
2.01 m
1.71 m
1.34 m
3.15 m
3.37 m
β
γ
σ
47.2
47.0
C=0
α
β
172.0
66.6
28.2
171.7
64.5
28.1
Pro1
4.94 d
2.31 m
1.80 m
2.05 m
1.96 m
4.01 m
3.40 m
5.03 Overlap
2.29 m
1.79 m
2.05 m
1.98 m
γ
25.1
47.6
172.4
25.0
47.4
172.2
σ
4.00 m
3.41 m
C=0
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SHAH et al./Turk J Chem
Table 2. QTOF/MS sequence ions (m/z) of the protonated molecular ions of cyclic peptide 1.
MS/MS
fragmentation of
[M+H+]
Proposed fragment structure
Pro1–Pro2–Ala3–Gly4–Leu5–Ala6–Thr7–Phe8
[M+H+]
775
737
608
[H–Pro1–Ala3–Gly4–Leu5–Ala6–Thr7]+
[(H–Pro1–Pro2–Ala3–Gly4–Leu5–Ala6–Thr7]-H2O]+ 590
[H–Pro1–Pro2–Ala3–Gly4–Leu5–Ala6]+
[H–Pro1–Pro2–Ala3–Gly4–Leu5]+
[H–Pro1–Pro2–Ala3]+
507
436
266
Table 3. Effect of brachystemin A (1) on production of inflammatory cytokines TNF-α, IL-1β , IL-2, and nitric oxide.
Effect of peptide on viability of 3T3 cells was also evaluated using MTT assay. The results are presented as mean ꢀ SD
of triplicates.
TNF-α
% Inh
IL-1β
% Inh
IL-2
% Inh
NO
% Inh
Cytotoxicity
IC50 (µM)
> 60
Compound
Brachystemin A
NG Methyl L-
Arginine Acetate
Cyclohexamide
19.3 ꢀ 1.0 7.5 ꢀ 1.8 35.2 ꢀ 11.4 7.4 ꢀ 0.1
-
-
-
65.65 ꢀ 1.1
-
-
-
-
-
0.1 ꢀ 0.2
In conclusion, the total synthesis of natural peptide brachystemin A (1), involving a solid-phase route
by using safety-catch linker, was carried out. The structure was identified by mass spectrometry and nuclear
magnetic resonance spectroscopy. Furthermore, this peptide was found to be nontoxic on a normal cell line
(3T3 fibroblast cells). In this study, brachystemin A was also found to be a moderate inhibitor of IL-2 and
TNF-α. Thus, it can be an important lead for drug discovery against inflammatory diseases. However, in vivo
studies are necessary to evaluate the effects of brachystemin A (1) in inflammatory diseases.
3. Experimental
3.1. General experimental procedures
Protected amino acids, resin, and all other chemicals and reagents were purchased from Sigma Aldrich, Chem-
impex, and Novabiochem. The peptide 1 was purified by RP-HPLC (LC-900 Japan). C18 Column Jaigel
ODS-MAT 80 was used in the purification of the peptide at a flow rate of 4 mL/min, and H2 O/CH3 OH (50:50)
was used a mobile phase. A Bruker 500 MHz was used for recording 1 H and 13 C nuclear magnetic resonance
spectra, and chemical shifts were reported in parts per million. Electron spray ionization mass spectra were
recorded on a QSTAR XL (Applied Biosystems).
3.2. Peptide synthesis
4-Sulfamylbutyryl AM resin was soaked in dimethylformamide (DMF) for 1 h. Fmoc-amino acid (4 equiv.) was
synthesized manually in a 10-mL polypropylene syringe fitted with a filter disc and agitation was performed on
an orbital shaker. PyBOP (2.28 g, 4 equiv.), and DIEA (1.5 mL, 8 equiv.) in DMF were added to the resin.
The reaction mixture was stirred for 24 h. This coupling step was repeated to achieve maximum loading.
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SHAH et al./Turk J Chem
3.3. Peptide coupling
The swollen peptidyl resin 3 was deprotected by 20% 4-methylpiperidine in DMF for 20 min. After the
deprotection step, the resin was washed with DMF. The next Fmoc-amino acid (3 equiv.) was activated
by PyBOP (4 equiv.), and DIEA (4 equiv.) in 5 mL of DMF.
3.4. Activation of sulfonamide linker
The Fmoc protecting group of linear peptidyl resin 4 was removed by 20% 4-methylpiperidine in DMF for
20 min, and the amino group of the terminal amino acid was protected by trityl chloride (1.227 g, 4 equiv.)
in the presence of DIEA (1.5 mL, 8 equiv.) for 2 h. The sulfonamide linker was activated by reaction with
iodoacetonitrile (0.8 mL, 10 equiv.) in the presence of DIEA (2.25 mL, 12 equiv.) and N -methylpyrrolidinone
(NMP) for 12 h under N2 . The reaction was protected from light by covering the reaction vessel with aluminum
foil. The resin was washed, and the trityl group was removed by 5% trifluoroacetic acid/dichloromethane for
2 h.
3.5. Cyclization and release of peptide from the resin
The activated N -acylsulfonamide linker was soaked in tetrahydrofuran and treated with base DIEA (565 µL,
3 equiv.) for 24 h under N2 . The resin was filtered and washed with tetrahydrofuran and dichloromethane (3
× 25 mL each). The filtrate was concentrated to remove solvents and the crude residue was precipitated with
cold diethyl ether. Finally, the side chain protecting groups were removed by treatment with TFA/TIS/H2 O
(9.5:0.25:0.25). The peptide residue was precipitated by cold ether, lyophilized, and then purified by RP-HPLC.
Brachystemin A (1) Cyclo-(Pro1 -Pro2 -Ala3 -Gly4 -Leu5 -Ala6 -Thr7 -Phe8); 59.5 mg (7.4%); [α]2D5 –23
(c0.0005, MeOH); 1 H NMR (500 MHz) and 13 C NMR (125-MHz) (Table 1); ESI-MS m/z 755 [M + H]+ ;
HR-TOF-ESI-MS m/z755.4068 [M + H]+ (calcd. for C37 H45 N8 O9 , 755.4092).
3.6. Nitrite concentration in mouse macrophage culture medium
The mouse macrophage cell line J774.2 (European Collection of Cell Cultures, UK) was cultured in 75-cc
flasks (IWAKI Asahi Techno Glass, Tokyo, Japan) in Dulbecco’s Modified Eagle’s Medium (Sigma-Aldrich,
Steinheim, Germany) that contained 10% fetal bovine serum (GIBCO, New York, NY, USA) supplemented
with 1% streptomycin/penicillin. The flasks were kept at 37 ◦ C in humidified air containing 5% CO2 . Cells
(106 cells/mL) were then transferred to a 24-well plate. The nitric oxide synthase (NOS-2) in macrophages was
induced by the addition of 30 µg/mL E. coli lipopolysaccharide (LPS) (Difco Laboratories, Detroit, MI, USA).
The test compounds were added at 30-µM concentration and cells were further incubated at 37 ◦ C in 5% CO2 .
The supernatant was collected after 48 h for analysis. Nitrite accumulation in cell culture supernatant was
measured using the Griess method described.27
3.7. Cytokine production and quantification
THP-1 (human monocytic leukemia cells) was obtained from the European Collection of Cell Cultures (UK).
The cells were maintained in endotoxin-free RPMI-1640 containing 5.5 mmol/L glucose (BioM Laboratories,
Chemical Division, Malaysia), 50 µmol/L mercaptoethanol (Merck, Darmstadt, Germany), 10% fetal bovine
936

SHAH et al./Turk J Chem
serum (FBS), 2 mmol/L L-glutamine (PAA Laboratories, GmbH, Pasching, Austria), 1 mmol/L sodium pyru-
vate (GIBCO, Grand Island, NY, USA), and 10 mmol/L (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid )
(MP Biomedicals, Illkirch, France). Cells were grown in 75-cc flasks until they attained 70% confluence, and
then were plated in 24-well tissue culture plates at a concentration of 2.5 × 105 cells/mL. The cells were differ-
entiated into macrophage-like cells by using phorbol myristate acetate (PMA) (SERVA, Heidelberg, Germany)
at a final concentration of 20 ng/mL and incubated for 24 h at 37 ◦ C in 5% CO2 . The cells were then stimulated
with E. coli Lipopolysacchride B (Difco Laboratories) at a final concentration of 50 ng/mL and treated with
peptide 1 at a concentration of 25 µM. The cells were then incubated for 4 h at 37 ◦ C in 5% CO2 . The super-
natants collected were analyzed for the level of TNF-αand IL-1β . Jurkat (human T lymphocyte leukemia) cells
were kindly provided by Prof Daniel Hoessli (University of Geneva, Switzerland). The cells were maintained in
RPMI-1640 supplemented with 5% FBS and 1% penicillin/streptomycin. Upon 70% confluence the cells were
plated in 96-well flat bottom plates at a concentration of 2 × 106 cells/mL. The cells were activated by using
20 ng/mL phorbol myristate acetate (PMA) and 7.5 µg/mL phytohemagglutinin (PHA) (SERVA, Heidelberg,
Germany). The cells were then treated with peptide 1 at a concentration of 25 µM and plate was incubated
for 18 h at 37 ◦ C in 5% CO2 . Supernatants were collected and analyzed for interleukin-2 cytokine. Cytokine
quantification in supernatants was performed using the human TNF-α, IL-1β , and IL-2 Kits Duo Set (R&D
Systems, Minneapolis, MN, USA), and according to the manufacturer’s instructions.
3.8. Cytotoxicity assay
An in vitro cytotoxicity assay was performed as described previously.28 Briefly 3T3 cells were harvested and
suspended in Dulbecco’s Modified Eagle’s Medium supplemented with 5% FBS. Then 100 µL of 6 × 104
cells/mL were plated in 96-well flat bottomed plates and the plates were incubated for 24 h at 37 ◦ C in 5%
CO2 . After incubation, media was carefully removed and the cells were charged with different concentrations
(10–100 µM) of cyclopeptide in triplicate; the final volume of 200 µL in each well was adjusted with complete
Dulbecco’s Modified Eagle’s Medium. Plates were then further incubated for 48 h at 37 ◦ C in a CO2 incubator.
After 48 h, the supernatant was carefully removed and 50 µL of 0.5 mg/mL (3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide) from 5 mg/mL stock was added to each well and the plates were then incubated
for a further 4 h. After incubation, (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was aspirated
and formazan crystals were dissolved by addition of 100 µL of dimethyl sulfoxide with gentle agitation for 10–15
min in an orbital shaker (MTS 2/4 Digital Microtiter Shaker, IKS, Staufen, Germany). The extent of (3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) reduction to formazan within cells was calculated by
measuring the absorbance at 540 nm, using a spectrophotometer. The cytotoxicity was recorded as concentration
causing 50% growth inhibition (IC50).
Acknowledgment
The author acknowledges the financial support from the Higher Education Commission, Pakistan (Grant #
20-1656/R & D/10).
Supporting Information
NMR, ESI-MS/MS spectra, analytical high performance liquid chromatography profile of peptide brachystemin
A.
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SHAH et al./Turk J Chem
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SHAH et al./Turk J Chem
Solid-phase total synthesis of cyclic peptide brachystemin A and its immunomodulating activity
Supporting Information
Zafar Ali SHAH, Almas JABEEN, Samreen SOOMRO, M. Ahmed MESAIK,
M. Iqbal CHOUDHARY, Farzana SHAHEEN
Figure S1
Figure S2
Figure S3
Figure S4
Figure S5
Figure S6
Figure S7
Figure S8
1H NMR spectrum of peptide (1) in d5-pyridine (300 MHz).
COSY NMR spectrum of peptide (1) in d5-pyridine (500 MHz).
HSQC NMR spectrum of peptide (1) in d5-pyridine (500 MHz).
HMBC NMR spectrum of peptide (1) in d5-pyridine (500 MHz).
HR-ESI mass spectra of peptide (1).
S2
S3
S4
S5
S6
S7
S8
S9
ESI mass spectra of peptide (1).
ESI MS/MS of peptide (1).
Analytical HPLC profile of peptide (1).
1

SHAH et al./Turk J Chem
Figure S1. 1 H NMR spectrum of peptide (1) in d5 -pyridine (300 MHz).
2

SHAH et al./Turk J Chem
Figure S2. COSY NMR spectrum of peptide (1) in d5 -pyridine (500 MHz).
3

SHAH et al./Turk J Chem
Figure S3. HSQC NMR spectrum of peptide (1) in d5 -pyridine (500 MHz).
4

SHAH et al./Turk J Chem
Figure S4. HMBC NMR spectrum of peptide (1) in d5 -pyridine (500 MHz).
5

SHAH et al./Turk J Chem
Figure S5. HR-ESI mass spectra of peptide (1).
6

SHAH et al./Turk J Chem
Figure S6. ESI mass spectra of peptide (1).
7

SHAH et al./Turk J Chem
Figure S7. ESI MS/MS of peptide (1).
8

SHAH et al./Turk J Chem
Figure S8. Analytical HPLC profile of peptide (1).
9