Authors
Keywords
Abstract
In this work, linear and cyclic peptides analogues of Longicalycinin A, known as a natural cyclopeptide anticancer agent, have been designed and successfully synthesized by solid phase peptide synthesis methodology of Fmoc/t-Bu. 2-Chlorotrityl chloride resin (2-CTC) was used as solid support. The synthesized linear (Ot-Bu) Longicalycinin A analogues were cleavaged by the method of partial cleavage for the separation of the solid phase. The final deprotection was performed by treatment with TFA 95% containing scavengers to achieve deprotected linear Longicalycinin A analogues. Macrocyclization of deprotected cyclic Longicalycinin A analogues were characterized by different instrumental methods using FT-IR, LC-MS, 1H-NMR and 13C-NMR. Deprotected linear and cyclic analogues, synthesized as such, were evaluated for their toxic activity against cell lines of HepG2 (human liver cancer cell line) and HT-29 (human colorectal adenocarcinoma cell line) using MTT assay. The synthetic linear and cyclic analogues showed relatively good activity against cell lines of HepG2 and HT-29 with IC50 values from 8.76 to 17.2 µg/mL and 9.06 to 17.34µg/mL, respectively, in comparison to standard drug 5-fluorouracil (5-FU). Safety profile of the synthesized liner and cyclic analogues of Longicalycinin A were also examined using skin Fibroblast cells. Among the linear peptides, linear compounds 1, 8, 12, 13 and 14, showed a considerable toxicity activity on cancer cell lines HT-29 than HepG2 along with a high safety on normal cells and among the cyclic peptides, compound 5, considering the property of toxicity action good enough on cancerous cell lines along with high safety profile on normal skin cells, can be good candidates for developing new anticancer agents
Introduction
Cancer is a disease marked by uncontrolled growth and division of cells, making tumors. If the tumor spread is not controlled, it can result in mortality. Cancer is caused by the both external factors (tobacco, chemicals, radiation, and infectious organisms) and internal factors (inherited mutations, hormones, immune conditions, and mutations that occur from metabolism).1Cancer can occur in the breast, lungs, colon, blood cells and etc. Cancers are similar in some ways, but different in growth and spread routes.2 Normal cells are divided in a regular routine order. They die when become damaged and new cells take their place.3 When cancer cells spread in the body, it is called metastasis. Many new and effective therapies are currently being used to treat cancer. Among the new ways to battle cancer, chemotherapy based on peptides has attracted a great interest and considerable attention. This is due to the unique advantages of peptides, such as having low molecular weights, the ability to specifically target tumor cells, and low toxicity in normal tissues. During the last decade, peptides have gained a wide range of applications in medicine, drug delivery and biotechnology.4
Peptides, a class of compounds consisting of two or more amino acids linked by peptide bond, and are abundantly present in living organisms. A large number of peptides have been isolated from animals, plants and microorganisms. Based on their chemical configuration, peptides can be divided into linear and cyclic peptides.5 Studies have shown that large numbers of peptides isolated from plants are cyclic peptides, so-called cyclopeptides. Cyclopeptides compared with linear peptides, exhibit more potent biological activities, possibly due to the stable scaffolds provided by their cyclic structure. Pharmacy studies have proved that many peptides, including those isolated from plants, have a potential antitumor effect.6-8 Peptides isolated from plants have a number of advantages over other chemical agents including relatively simple structure, their low molecular weight, lower antigenicity and fewer adverse actions, easy absorption, and a variety of routes of administration.9 Recently, one of the most active areas of research is the search for natural components of plants with potent antitumor activity and low toxicity.10 The inherent medicinal properties of cyclic peptides promoted scientists to isolate these compounds from natural sources. There are four possible ways that a peptide can be constrained as a macro cycle: head-to-tail (C-terminus to N-terminus), head-to-side chain, side chain-to-head, or side chain-to-side chain.11 Cyclic peptides head to tail can easily be synthesized in good purity using standard procedures. However, the cyclization strategy is critical and depends upon the sequence of peptide, structural constraints, and the resulting ring size. A number of studies have been developed to improve this crucial strategy and to obtain cyclic peptides in good yield with minimum side reactions.12
One of the preferred methods for peptide synthesis is the synthesis on solid phase. Among the various resins employed for the synthesis of peptides is 2-chlorotrityl chloride resin. The trityl linker allows the protected compounds to be subjected to various chemical manipulations and consequently to afford pure compounds without employing numerous purification steps.13-17 Oshima et al isolated a cyclic pentapeptide Longicalycinin A [cyclo-(Gly-Phe-Pro-Tyr-Phe)] from the plant Dianthus superbus var. Longicalycinus which has been used for treating carcinoma, diuretic and inflammatory diseases.18-21 The aim of this study was to design and synthesize linear and cyclic pentapeptides analogues of Longicalycinin A that might have been expected to produce anticancer activity. The cytotoxic activities of synthesized peptides were evaluated against various human cancer cell lines including, HepG2 and HT-29.
Experimental:
Materials and Instruments
All other commercially obtained reagents and solvents were used without further purification. Trifluoroacetic acid (TFA), Fmoc amino acids and coupling reagents O-(7-Azabenzotriazol-1-yl)-N,N,N,N-tetramethyluronium hexafluorophosphate (HATU), (benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyBop), Solvents like acetonitrile (MeCN), Piperazine, N,N-diisopropylethylamine (DIPEA), Diethylether, Dichloromethane (DCM), N,N-dimethylformamide (DMF), and methanol (MeOH) were purchased from Merck Co. Germany. 2-chlorotritylchloride resin (1% DVB, 200-400 mesh, 1 mmol/g) was purchased from Aldrich, Switzerland. Commercially available chemicals were used without further purification unless otherwise stated. Flash column chromatography was carried out using silica Gel 60 (particle size 0.04–0.06 mm / 230–400 mesh). FTIR spectra were obtained on SHIMADZU recording and Nicolet Magna 550 spectrometers. The mass spectral measurements were performed on a 6410 Agilent LC-MS triple quadruple mass spectrometer with an electrospray ionization (ESI) interface, USA. 1H-NMR and 13C-NMR spectra were obtained with a bruker 300 MHz spectrometer.
Designing linear peptides of Longicalycinin A analogues
We designed different amino acids for each position of diversities R1, R2, R3, R4 and R5 for the synthesis of eighteen Longicalycinin A analogues, based on conserving lipophilic characteristic of peptides (1-18). Thus, we chose Ala, Val and Gly for position R1, Phe and Ala for position R2, Pro for position R3, Tyr (t-Bu), Ser(t-Bu) and Thr(t-Bu) for position R4 and Gly, Leu and Phe for position R5 in the first category. Ala, Leu and Val for position R1, Phe and Ile for position R2, Val, Pro, Gly and Phe for position R3, Tyr(t-Bu) and Thr(t-Bu) for position R4 and Pro, Thr(t-Bu) and Val for position R5 in the second category. Gly, Val, Ala and Ile for position R1, Phe and Ala for position R2, Pro for position R3, Tyr(t-Bu), Ser(t-Bu) for position R4 and Ala and Phe for position R5 in the third category.
General procedure for the synthesis of linear pentapeptides (O t -Bu) Longicalycinin A analogues (Schemes 1 and 2)
The syntheses of linear pentapeptides (Ot-Bu) Longicalycinin A analogues were carried out using 2-chlorotrityl chloride resin (1 mmol/g) following the standard Fmoc strategy. The first amino acid Fmoc-Val-OH (680 mg, 2 mmol) was attached to the 2-CTC resin using di isopropyl ethyl amine (DIPEA) (1 mL) in anhydrous di chloro methane and N, N- di methyl formamide (DCM:DMF) (30 mL, 1:1) at room temperature for 2h. After filtration, the remained tritylchloride groups were capped by a solution of DCM / CH3OH / DIPEA (3:2.5:1.2) for 30 min. The mixture was filtered and washed thoroughly with DCM (10 mL) and DMF (2 × 20 mL). The resin-bound Fmoc-amino acid was treated with Piperazine 10% in DMF (100 mL) for 30 min and the resin was washed with DMF (4 × 20 mL). A solution of the second amino acid Fmoc-Phe-OH (780 mg, 2.01 mmol), HATU (650 mg, 1.7 mmol), and DIPEA (0.5 mL) in 10 mL DCM were added to the resin-bound free amine and shaken for 2h at room temperature. After completion of the coupling reaction, the resin was washed with DMF (2 × 10 mL). The resin-bound Fmoc-peptide was treated with Piperazine 10% in DMF (100 mL) for 30 min and the resin was washed with DMF (4 × 20 mL). Other protected amino acids, i.e., Fmoc-Pro-OH, Fmoc-Tyr(t-Bu)-OH and Fmoc-Gly-OH were added to the resin-peptide, sequentially, with the same procedure mentioned as above. In the all cases for detecting the presence or absence of free primary amino groups on the resin-peptide, chloranil test was used. The produced pentapeptide was cleaved from the resin by treatment of TFA 1% in DCM and neutralized with pyridine 4% in CH3OH. The solvent was removed under reduced pressure and the residue was precipitated in water. The precipitate was filtered and dried. Other analogues were synthesized in the same way.
Scheme 1. The Solid-Phase Synthesis of Longicalycinin A analogue (1)via 2-chlorotrityl chloride resin
Scheme 2. The Solid-Phase Synthesis of protected Longicalycinin A analogue (1)
General procedure for the synthesis of deprotected linear Longicalycinin A analogues (Scheme 3)
A mixture of trifluoroacetic acid (1 mL), dichloromethane (1 mL), anisole (10) and phenol (0.2 g) were added to the protected linear pentapeptides and stirred for 1h. Under such strong acidic condition, cleavage of side chain protecting groups of the peptides was achieved. Then, the excess TFA / DCM were removed under reduced pressure. The desired pentapeptides were precipitated in cold diethyl ether and collected by filtration.
Scheme 3. The Solid-Phase Synthesis of deprotected Longicalycinin A analogue (1)
General procedure for the synthesis of cyclic Longicalycinin A analogues (scheme 4)
All the Precipitates of linear (Ot-Bu) Longicalycinin A analogues were dissolved in CH3CN (100 mL) and treated with PyBop (2 eq) and DIPEA (4 eq). Obtained cyclic peptides were in good yield with minimum side reactions. Cyclic peptides were achieved by column chromatography (4:1, chloroform: methanol). Final deprotection on cyclic (Ot-Bu) LongicalycininA analogues were done by treatment of TFA 95%, phenol and anisole as scavengers. The excess TFA / DCM were removed under reduced pressure. The desired peptides were precipitated in cold diethylether. The other cyclic analogues were synthesized in the same way.
Scheme 4. The Solid-Phase Synthesis of protected and deprotected cyclic Longicalycinin A analoguevia2-chlorotritylChloride resin
MTT assay
To determine the cytotoxicity of the linear and cyclic Longicalycinin A analogues, two human tumor cell lines were used, HepG2 and HT-29. Skin Fibroblast cell line was chosen as a safety control for normal cells. For the measurement of cell viability, succinate dehydrogenase (SDH) activity was assessed using the MTT test after 6h incubation of the cells with Longicalycinin A analogues (1.25, 2.5, 5, 10, 20, 50 and 100 µg/mL). The above-mentioned cells were cultured in RPMI1640 medium at 37 under 5% CO2, 95% air, supplemented with 10% fetal bovine serum (FBS), 100U/mL penicillin and 100µg/mL streptomycin. The cells were seeded into 96-well plates with the concentration of 104cells/well and allowed to grow for 24h and then incubated with the increasing concentrations of each peptide compound for 6h. Cell activity was analyzed by using a MTT method which is based on the conversion of 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) orange dye to purple formazan crystals by mitochondrial succinate dehydrogenase enzyme in living cells. At the end of each treatment period, MTT dye (10, 5 mg/mL in phosphate-buffered saline) was added to each well and the micro plate was incubated at 37 for 4h. The medium containing MTT was removed and DMSO (10 was added to each well to dissolve the formazan crystals. The plate was shaken for 30 min at 37 and the absorbance was read at 570 nm using a spectrophotometer plate reader (Infinite® M200, TECAN).22 5-Fluorouracil was also used as a positive control and DMSO solvent as the blank for the test compounds. Data were presented as the mean of triplicate measurement of the number of living cells and their capacity to reduce MTT reagent. IC50 values were calculated by using Prism software.
Cell culture for LDH assay
For LDH assay, the HepG2 and HT-29 cells were maintained in cell culture flasks (75 cm2) with RPMI 1640 cell culture medium containing 10% fetal bovine serum (FBS) in an incubator (37 °C, 5% CO2, 95% air) and were passaged every 4 or 5 days. To passage cells, the cell culture flasks were washed with phosphate-buffered saline (PBS) three times and trypsin was added to detach cells from the bottom of the flasks.
LDH assay
LDH catalyzes the conversion of lactate to pyruvate, the forward reaction and the conversion of pyruvate to lactate, the reverse reaction. Lactate and NAD+ are converted to pyruvate and NADH by the action of LDH. NADH strongly absorbs light at 340 nm, whereas NAD+ does not. The rate of increase in absorbance at 340 nm is directly proportional to the LDH activity in the sample.23 Cytotoxicity induced by peptides was assessed by lactate dehydrogenase (LDH) leakage into the culture medium. The activity of LDH in the medium was determined using a commercially available kit from Sigma-Aldrich. To prepare samples for the LDH assay, the cancerous cells of passage numbers 11-20 were used. The cell samples (1mL, at a density of 107 cells/mL RPMI containing 10% FBS) were seeded in each well of 12-well plates and grown for 24hr before exposure to linear and cyclic Longicalycinin A peptide analogues. The cells were washed with PBS three times. Cell samples dosed with 10 µg/mL concentration of Longicalycinin A analogues for 6hr exposure in RPMI medium containing 1% FBS. The 12-well plates were shaken briefly to homogenize the released LDH in the cell culture medium and the medium was transferred to 1.5mL-microcentrifuge tubes and centrifuged at 1,000 ×g at 4°C for 15 min to remove any cell debris. The supernatant was separated and the absorbance at 340 nm was measured using ELISA reader. 24,25 The LDH assay of the samples was obtained by measuring optical density.
Flow cytometry analysis and Identification of apoptosis by PI staining
Flow cytometry analysis was determined by analytical DNA flow cytometry. In this work, HepG2 and HT-29 cells were harvested and adjusted to 104cells/mL (SPL, Korea) and then incubated for 6h with 10µg/mL of peptide samples. The cells were centrifuged at high speed (12,000 rpm) for 20 s. The pellet was washed with saline buffer, after a new centrifugation, resuspended in 0.2 mL of lysis buffer (0.1% sodium citrate and 0.1% Triton X-100) containing 50 ?g/mL propidium iodide (PI) and stained with this reagent, a highly water-soluble fluorescent compound, at 37 °C for 15 min in the dark. The cells were then evaluated for the DNA fragmentation analysis using a FACScalibur flow cytometry equipment (Becton Dickinson, CA, USA) supplied with the flowing software 2.5.1.26
Determination of Lysosomal membrane integrity assay on HepG2 and HT-29
Lysosome damage using acridine orange (AO) as a probe was assayed in isolated lysosome obtained following HepG2 and HT-29 incubation with the peptide’s samples. Aliquots of the cell suspension (0.5 mL) previously incubated with the peptide’s samples (10 µg/mL) were stained withAO (5 µM) and precipitated from the incubation medium by 1 min centrifugation at 1000 rpm. The cell pellet was then suspended in 2 mL fresh incubation medium. This washing process was carried out twice to remove the fluorescent dye from the media. The AO redistribution in the cell suspension was then measured fluorimetrically using a Shimadzu RF5000U fluorescence spectrophotometer set at 495 nm excitation and 530 nm emission wavelengths. Lysosomal membrane damage was determined as the difference in redistribution of acridine orange from lysosomes into cytosol between treated cells and control cells at the time of preparation. 27,28
Statistical analysis
Analyses were performed using GraphPad Prism 5 (GraphPad Software, La Jolla, CA, USA). Statistical analysis among groups was performed using multiple comparisons by one way ANOVA followed by Tukey’s post hoctest. All data are presented as arithmetic mean ± S.E.M of at least triplicate determinations. Significance was accepted at P<0.05.
Results:
The protected and deprotected linear Longicalycinin A analogues
Synthesis of Gly-Tyr-Pro-Phe-Val (1)
a) Synthesis of protected peptide
Yield: 75%; Yellow oily liquid; FTIR (KBr): (cm-1) 3407 (NH amide), broad peak 2400-3400 (OH COOH), 1677 (C=O COOH), 1641 (C=O amide), 1544 (C=C in amino acids Phenylalanine and Tyrosine), 1197 (C-O COOH), 1134 (C-O), 600-800 (out of plane bending vibration C-H in amino acids Phenylalanine and Tyrosine); LC-MS (ESI) m/z Calcd for (1a) 637.35, Found m/z = 635.9 (M-1), m/z = 638.3 (M+1).
b) Synthesis of deprotected peptide
Yield: 75%; White solid; FTIR (KBr): (cm-1) 3461.23 (OH), C=O amide under 1668.21 (C=O COOH) is also buried, 1546.85 (C=C in amino acids Phenylalanine and Tyrosine), 1192.63 (C-O COOH), 1131.96 (C-O), 600-800 (out of plane bending vibration C-H in amino acids Phenylalanine and Tyrosine); LC-MS (ESI) m/z Calcd for (1b) 581.29, Found m/z = 580.1 (M-1), m/z = 582.8 (M+1).
Synthesis of Leu-Tyr-Pro-Phe-Ala (2)
a) Synthesis of protected peptide
Yield: 77%; Yellow oily liquid; FTIR (KBr): (cm-1) 3407 (NH amide), broad peak 2400-3400 (OH COOH), 1677.95 (C=O COOH), 1641.31 (C=O amide), 1541.02 (C=C in amino acids Phenylalanine and Tyrosine), 1199.64 (C-O COOH), 1137.92 (C-O), 600-800 (out of plane bending vibration C-H in amino acids Phenylalanine and Tyrosine); LC-MS (ESI) m/z Calcd for (2a) 665.38, Found m/z = 663.9 (M-1), m/z = 666.3 (M+1).
b) Synthesis of deprotected peptide
Yield: 77%; White solid; FTIR (KBr): (cm-1) 3461.23 (OH), C=O amide under 1670.13 (C=O COOH) is also buried, 1546.90 (C=C in amino acids Phenylalanine and Tyrosine), 1194.19 (C-O COOH), 1133.72 (C-O), 600-800 (out of plane bending vibration C-H in amino acids Phenylalanine and Tyrosine); LC-MS (ESI) m/z Calcd for (2b) 609.32, Found m/z = 608.9 (M-1), m/z = 610.8 (M+1).
ynthesis of Gly-Tyr-Pro-Phe-Ala (3) S
a) Synthesis of protected peptide
Yield: 78%; Yellow oily liquid; FTIR (KBr): IR (KBr): (cm-1) 3407 (NH amide), broad peak 2400-3400 (OH COOH), 1672.17 (C=O COOH), 1641.31 (C=O amide), 1546.80 (C=C in amino acids Phenylalanine and Tyrosine), 1197.71 (C-O COOH), 1134.07 (C-O), 600-800 (out of plane bending vibration C-H in amino acids Phenylalanine and Tyrosine); LC-MS (ESI) m/z Calcd for (3a) 609.32, Found m/z = 607.9 (M-1), m/z = 610.2 (M+1).
b) Synthesis of deprotected peptide
Yield: 78%; White solid; FTIR (KBr): (cm-1) 3461.23 (OH), 3278.32 (NH), C=O amide under 1672.77 (C=O COOH) is also buried, 1517.18 (C=C in amino acids Phenylalanine and Tyrosine), 1199.43 (C-O COOH), 1135.08 (C-O), 600-800 (out of plane bending vibration C-H in amino acids Phenylalanine and Tyrosine); LC-MS (ESI) m/z Calcd for (3b) 553.26, Found m/z = 551.9 (M-1), m/z = 554.7 (M+1).
of Ala-Tyr-Pro-Phe-Gly (4)Synthesis
a) Synthesis of protected peptide
Yield: 80%; Yellow oily liquid; FTIR (KBr): (cm-1) 3407 (NH amide), broad peak 2400-3400 (OH COOH), 1676.03 (C=O COOH), 1641.31 (C=O amide), 1546.80 (C=C in amino acids Phenylalanine and Tyrosine), 1201.57 (C-O COOH), 1135.99 (C-O), 600-800 (out of plane bending vibration C-H in amino acids Phenylalanine and Tyrosine); LC-MS (ESI) m/z Calcd for (4a) 609.32, Found m/z = 608.1 (M-1), m/z = 610.2 (M+1).
b) Synthesis of deprotected peptide
Yield: 80%; White solid; FTIR (KBr): (cm-1) 3461.23 (OH), C=O amide under 1667.76 (C=O COOH) is also buried, 1546.88 (C=C in amino acids Phenylalanine and Tyrosine), 1194.60 (C-O COOH), 1132.67 (C-O), 600-800 (out of plane bending vibration C-H in amino acids Phenylalanine and Tyrosine); LC-MS (ESI) m/z Calcd for (4b) 553.26, Found m/z = 551.9 (M-1), m/z = 554.8(M+1).
Synthesis ofPhe-Ser-Pro-Phe-Gly(5)
a) Synthesis of protected peptide
Yield: 80%; Yellow oily liquid; FTIR (KBr): (cm-1) 3407 (NH amide), broad peak 2400-3400 (OH COOH), 1668.31 (C=O COOH), 1641.31 (C=O amide), 1546.80 (C=C in amino acids Phenylalanine), 1195.78 (C-O COOH), 1134.07 (C-O), 600-800 (out of plane bending vibration C-H in amino acids Phenylalanine); LC-MS (ESI) m/z Calcd for (5a) 609.32, Found m/z = 608.0 (M-1), m/z = 610.2 (M+1).
b) Synthesis of deprotected peptide
Yield: 80%; White solid FTIR; IR (KBr): (cm-1) 3499.87 (OH), C=O amide under 1673.86 (C=O COOH) is also buried, 1548.08 (C=C in amino acids Phenylalanine), 1194.60 (C-O COOH), 1130.81 (C-O), 600-800 (out of plane bending vibration C-H in amino acids Phenylalanine); LC-MS (ESI) m/z Calcd for (5b) 553.26, Found m/z = 551.9 (M-1), m/z = 554.3 (M+1).
Synthesis ofVal-Tyr-Pro-Ile-Ala (6)
a) Synthesis of protected peptide
Yield: 74%; Yellow oily liquid; FTIR (KBr): (cm-1) 3407 (NH amide), broad peak 2400-3400 (OH COOH), 1670.24 (C=O COOH), 1641.31 (C=O amide), 1546.80 (C=C in amino acid Tyrosine), 1197.71 (C-O COOH), 1132.14 (C-O), 600-800 (out of plane bending vibration C-H in amino acid Tyrosine); LC-MS (ESI) m/z Calcd for (6a) 617.38, Found m/z = 616.1 (M-1), m/z = 618.3 (M+1).
b) Synthesis of deprotected peptide
Yield: 74%; White solid; FTIR (KBr): (cm-1) 3474.79 (OH), C=O amide under 1670.32 (C=O COOH) is also buried, 1546.10 (C=C in amino acid Tyrosine), 1191.37 (C-O COOH), 1132.54 (C-O), 600-800 (out of plane bending vibration C-H in amino acid Tyrosine); LC-MS (ESI) m/z Calcd for (6b) 561.32, Found m/z = 560.2 (M-1), m/z = 562.5 (M+1).
Synthesis ofPro-Tyr-Val-Phe-Ala (7)
a) Synthesis of protected peptide
Yield: 77%; Yellow oily liquid; FTIR (KBr): (cm-1) 3407 (NH amide), broad peak 2400-3400 (OH COOH), 1679.88 (C=O COOH), 1641.31 (C=O amide), 1546.80 (C=C in amino acids Phenylalanine and Tyrosine), 1203.50 (C-O COOH), 1135.99 (C-O), 600-800 (out of plane bending vibration C-H in amino acids Phenylalanine and Tyrosine); LC-MS (ESI) m/z Calcd for (7a) 651.37, Found m/z = 650.0 (M-1), m/z = 652.3 (M+1).
b) Synthesis of deprotected peptide
Yield: 77%; White solid; FTIR (KBr): (cm-1) 3445.20 (OH), C=O amide under 1676.59 (C=O COOH) is also buried, 1547.59 (C=C in amino acids Phenylalanine and Tyrosine), 1198.25 (C-O COOH), 1135.86 (C-O), 600-800 (out of plane bending vibration C-H in amino acids Phenylalanine and Tyrosine); LC-MS (ESI) m/z Calcd for (7b) 595.3, Found m/z = 594.9 (M-1), m/z = 596.8 (M+1).
Synthesis of Phe-Tyr-Pro-Val-Gly(8)
a) Synthesis of protected peptide
Yield: 79%; Yellow oily liquid; FTIR (KBr): (cm-1) 3407 (NH amide), broad peak 2400-3400 (OH COOH), 1679.88 (C=O COOH), 1641.31 (C=O amide), 1554.52 (C=C in amino acids Phenylalanine and Tyrosine), 1199.64 (C-O COOH), 1132.14 (C-O), 600-800 (out of plane bending vibration C-H in amino acids Phenylalanine and Tyrosine); LC-MS (ESI) m/z Calcd for (8a) 637.35, Found m/z = 635.9 (M-1), m/z = 638.3 (M+1).
b) Synthesis of deprotected peptide
Yield: 79%; White solid; FTIR (KBr): (cm-1) 3454.09 (OH), C=O amide under 1674.74 (C=O COOH) is also buried, 1547.55 (C=C in amino acids Phenylalanine and Tyrosine), 1194.60 (C-O COOH), 1132.42 (C-O), 600-800 (out of plane bending vibration C-H in amino acids Phenylalanine and Tyrosine); LC-MS (ESI) m/z Calcd for (8b) 581.29, Found m/z = 580.9 (M-1), m/z = 582.7 (M+1).
Synthesis ofPhe-Tyr-Pro-Phe-Val (9)
a) Synthesis of protected peptide
Yield: 75%; Yellow oily liquid; FTIR (KBr): (cm-1) 3407 (NH amide), broad peak 2400-3400 (OH COOH), 1681.81 (C=O COOH), 1641.31 (C=O amide), 1541.02 (C=C in amino acids Phenylalanine and Tyrosine), 1203.50 (C-O COOH), 1135.99 (C-O), 600-800 (out of plane bending vibration C-H in amino acids Phenylalanine and Tyrosine); LC-MS (ESI) m/z Calcd for (9a) 727.4, Found m/z = 726.0 (M-1), m/z = 728.3 (M+1).
b) Synthesis of deprotected peptide
Yield: 75%; White solid; FTIR (KBr): (cm-1) 3461.23 (OH), C=O amide under 1663.79 (C=O COOH) is also buried, 1547.13 (C=C in amino acids Phenylalanine and Tyrosine), 1194.60 (C-O COOH), 1129.62 (C-O), 600-800 (out of plane bending vibration C-H in amino acids Phenylalanine and Tyrosine); LC-MS (ESI) m/z Calcd for (9b) 671.33, Found m/z = 669.9 (M-1), m/z = 672.5 (M+1).
Synthesis of Phe-Tyr-Pro-Phe-Ala (10)
a) Synthesis of protected peptide
Yield: 75%; Yellow oily liquid; FTIR (KBr): (cm-1) 3407 (NH amide), broad peak 2400-3400 (OH COOH), 1677.95 (C=O COOH), 1641.31 (C=O amide), 1544.88 (C=C in amino acids Phenylalanine and Tyrosine), 1203.50 (C-O COOH), 1135.99 (C-O), 600-800 (out of plane bending vibration C-H in amino acids Phenylalanine and Tyrosine); LC-MS (ESI) m/z Calcd for (10a) 699.37, Found m/z = 697.9 (M-1), m/z = 700.3 (M+1).
b) Synthesis of deprotected peptide
Yield: 75%; White solid; FTIR (KBr): (cm-1) 3467.04 (OH), C=O amide under 1669.47 (C=O COOH) is also buried, 1547.25 (C=C in amino acids Phenylalanine and Tyrosine), 1196.40 (C-O COOH), 1132.62 (C-O), 600-800 (out of plane bending vibration C-H in amino acids Phenylalanine and Tyrosine); LC-MS (ESI) m/z Calcd for (10b) 643.3, Found m/z = 642.9 (M-1), m/z = 644.9 (M+1).
Synthesis of Thr-Val-Pro-Phe-Ala (11)
a) Synthesis of protected peptide
Yield: 76%; Yellow oily liquid; FTIR (KBr): (cm-1) 3407 (NH amide), broad peak 2400-3400 (OH COOH), 1681.81 (C=O COOH), 1641.31 (C=O amide), 1541.02 (C=C in amino acid Phenylalanine), 1203.50 (C-O COOH), 1135.99 (C-O), 600-800 (out of plane bending vibration C-H in amino acid Phenylalanine); LC-MS (ESI) m/z Calcd for (11a) 589.35, Found m/z = 588.0 (M-1), m/z = 590.3 (M+1).
b) Synthesis of deprotected peptide
Yield: 76%; White solid; FTIR (KBr): (cm-1) 3461.96 (OH), C=O amide under 1670.34 (C=O COOH) is also buried, 1547.07 (C=C in amino acid Phenylalanine), 1195.90 (C-O COOH), 1132.35 (C-O), 600-800 (out of plane bending vibration C-H in amino acid Phenylalanine); LC-MS (ESI) m/z Calcd for (11b) 533.29, Found m/z = 531.9 (M-1), m/z = 534.5 (M+1).
Synthesis of Phe-Tyr-Pro-Phe-Ile (12)
a) Synthesis of protected peptide
Yield: 78%; Yellow oily liquid; FTIR (KBr): (cm-1) 3407 (NH amide), broad peak 2400-3400 (OH COOH), 1681.81 (C=O COOH), 1641.31 (C=O amide), 1541.02 (C=C in amino acids Phenylalanine), 1203.50 (C-O COOH), 1137.92 (C-O), 600-800 (out of plane bending vibration C-H in amino acids Phenylalanine); LC-MS (ESI) m/z Calcd for (12a) 741.41, Found m/z = 740.0 (M-1), m/z = 742.3 (M+1).
b) Synthesis of deprotected peptide
Yield: 78%; White solid; FTIR (KBr): (cm-1) 3493.81 (OH), C=O amide under 1674.28 (C=O COOH) is also buried, 1546.97 (C=C in amino acids Phenylalanine and Tyrosine), 1196.18 (C-O COOH), 1133.05 (C-O), 600-800 (out of plane bending vibration C-H in amino acids Phenylalanine and Tyrosine); LC-MS (ESI) m/z Calcd for (12b) 685.35, Found m/z = 683.9 (M-1), m/z = 686.4 (M+1).
Synthesis of Val-Thr-Pro-Phe-Leu (13)
a) Synthesis of protected peptide
Yield: 80%; Yellow oily liquid; FTIR (KBr): (cm-1) 3407 (NH amide), broad peak 2400-3400 (OH COOH), 1679.88 (C=O COOH), 1645.17 (C=O amide), 1542.95 (C=C in amino acid Phenylalanine), 1203.50 (C-O COOH), 1135.99 (C-O), 600-800 (out of plane bending vibration C-H in amino acid Phenylalanine); LC-MS (ESI) m/z Calcd for (13a) 631.4, Found m/z = 630.0 (M-1), m/z = 632.3 (M+1).
b) Synthesis of deprotected peptide
Yield: 80%; White solid; FTIR (KBr): (cm-1) 3461.23 (OH), C=O amide under 1670.07 (C=O COOH) is also buried, 1547.28 (C=C in amino acid Phenylalanine), 1194.60 (C-O COOH), 1132.01 (C-O), 600-800 (out of plane bending vibration C-H in amino acid Phenylalanine); LC-MS (ESI) m/z Calcd for (13b) 575.33, Found m/z = 573.9 (M-1), m/z = 576.5 (M+1).
Synthesis of Pro-Tyr-Phe-Phe-Leu (14)
a) Synthesis of protected peptide
Yield: 72%; Yellow oily liquid; FTIR (KBr): (cm-1) 3407 (NH amide), broad peak 2400-3400 (OH COOH), 1679.88 (C=O COOH); 1637.45 (C=O amide), 1542.95 (C=C in amino acids Phenylalanine and Tyrosine), 1203.50 (C-O COOH), 1135.99 (C-O), 600-800 (out of plane bending vibration C-H in amino acids Phenylalanine and Tyrosine); LC-MS (ESI) m/z Calcd for (14a) 741.41, Found m/z = 740.0 (M-1), m/z = 742.4 (M+1).
b) Synthesis of deprotected peptide
Yield: 72%; White solid; FTIR (KBr): (cm-1) 3463.81 (OH), C=O amide under 1669.83 (C=O COOH) is also buried, 1546.08 (C=C in amino acids Phenylalanine and Tyrosine), 1191.50 (C-O COOH), 1132.97 (C-O), 600-800 (out of plane bending vibration C-H in amino acids Phenylalanine and Tyrosine); LC-MS (ESI) m/z Calcd for (14b) 685.35, Found m/z = 684.5 (M-1), m/z = 686.5 (M+1).
Synthesis of Pro-Tyr-Gly-Phe-Val (15)
a) Synthesis of protected peptide
Yield: 73%; Yellow oily liquid; FTIR (KBr): (cm-1) 3407 (NH amide), broad peak 2400-3400 (OH COOH), 1679.88 (C=O COOH), 1641.31 (C=O amide), 1542.95 (C=C in amino acids Phenylalanine and Tyrosine), 1203.50 (C-O COOH), 1137.92 (C-O), 600-800 (out of plane bending vibration C-H in amino acids Phenylalanine and Tyrosine); LC-MS (ESI) m/z Calcd for (15a) 637.35, Found m/z = 636.0 (M-1), m/z = 638.3 (M+1).
b) Synthesis of deprotected peptide
Yield: 73%; White solid; FTIR (KBr): (cm-1) 3461.23 (OH), 3284.38 (NH amide), 1676.24 (C=O COOH), 1638.33 (C=O amide), 1546.48 (C=C in amino acids Phenylalanine and Tyrosine), 1200.21 (C-O COOH), 1133.74 (C-O), 600-800 (out of plane bending vibration C-H in amino acids Phenylalanine and Tyrosine); LC-MS (ESI) m/z Calcd for (15b) 581.29, Found m/z = 580.6 (M-1), m/z = 582.7 (M+1).
Synthesis of Phe-Thr-Pro-Phe-Val (16)
a) Synthesis of protected peptide
Yield: 76%; Yellow oily liquid; FTIR (KBr): (cm-1) 3407 (NH amide), broad peak 2400-3400 (OH COOH), 1679.88 (C=O COOH), 1641.31 (C=O amide), 1541.02 (C=C in amino acids Phenylalanine), 1201.57 (C-O COOH), 1135.99 (C-O), 600-800 (out of plane bending vibration C-H in amino acids Phenylalanine); LC-MS (ESI) m/z Calcd for (16a) 665.38, Found m/z = 664.0 (M-1), m/z = 666.2 (M+1).
b) Synthesis of deprotected peptide
Yield: 76%; White solid; FTIR (KBr): (cm-1) 3307.44 (OH), C=O amide under 1672.15 (C=O COOH) is also buried, 1535.11 (C=C in amino acids Phenylalanine), 1200.56 (C-O COOH), 1137.61 (C-O), 600-800 (out of plane bending vibration C-H in amino acids Phenylalanine); LC-MS (ESI) m/z Calcd for (16b) 609.32, Found m/z = 608.1 (M-1), m/z = 610.1 (M+1).
Synthesis of Phe-Ser-Pro-Phe-Ala (17)
a) Synthesis of protected peptide
Yield: 77%; Yellow oily liquid; FTIR (KBr): (cm-1) 3407 (NH amide), broad peak 2400-3400 (OH COOH), 1674.10 (C=O COOH), 1643.24 (C=O amide), 1541.02 (C=C in amino acids Phenylalanine), 1199.64 (C-O COOH), 1134.07 (C-O), 600-800 (out of plane bending vibration C-H in amino acids Phenylalanine); LC-MS (ESI) m/z Calcd for (17a) 623.33, Found m/z = 621.9 (M-1), m/z = 624.3 (M+1).
b) Synthesis of deprotected peptide
Yield: 77%; White solid; FTIR (KBr): (cm-1) 3471.19 (OH), C=O amide under 1667.57 (C=O COOH) is also buried, 1547.01 (C=C in amino acids Phenylalanine), 1195.24 (C-O COOH), 1132.36 (C-O), 600-800 (out of plane bending vibration C-H in amino acids Phenylalanine); LC-MS (ESI) m/z Calcd for (17b) 567.27, Found m/z = 565.9 (M-1), m/z = 568.7 (M+1).
Synthesis of Phe-Ser-Pro-Ala-Gly (18)
a) Synthesis of protected peptide
Yield: 76%; Yellow oily liquid; FTIR (KBr): (cm-1) 3407 (NH amide), broad peak 2400-3400 (OH COOH), 1676.03 (C=O COOH), 1633.59 (C=O amide), 1546.80 (C=C in amino acid Phenylalanine), 1203.50 (C-O COOH), 1135.99 (C-O), 600-800 (out of plane bending vibration C-H in amino acid Phenylalanine); LC-MS (ESI) m/z Calcd for (18a) 533.29, Found m/z = 532.0 (M-1), m/z = 534.2 (M+1).
b) Synthesis of deprotected peptide
Yield: 76%; White solid; FTIR (KBr): (cm-1) 3309.47 (OH), C=O amide under 1673.18 (C=O COOH) is also buried, 1540.80 (C=C in amino acid Phenylalanine), 1199.65 (C-O COOH), 1136.13 (C-O), 600-800 (out of plane bending vibration C-H in amino acid Phenylalanine); LC-MS (ESI) m/z Calcd for (18b) 477.22, Found m/z = 475.9 (M-1), m/z = 478.1 (M+1).
Synthesis of Phe-Tyr-Pro-Phe-Gly (Linear Longicalycinin A) (19)
a) Synthesis of protected peptide
Yield: 78%; Yellow oily liquid; FTIR (KBr): (cm-1) 3407 (NH amide), broad peak 2400-3400 (OH COOH), 1676.03 (C=O COOH), 1641.31 (C=O amide), 1542.95 (C=C in amino acids Phenylalanine and Tyrosine), 1199.64 (C-O COOH), 1134.07 (C-O), 600-800 (out of plane bending vibration C-H in amino acids Phenylalanine and Tyrosine); LC-MS (ESI) m/z Calcd for (19a) 685.35, Found m/z = 684.0 (M-1), m/z = 686.3 (M+1).
b) Synthesis of deprotected peptide
Yield: 78%; White solid; FTIR (KBr): (cm-1) 3323.98 (OH), C=O amide under 1673.08 (C=O COOH) is also buried, 1540.36 (C=C in amino acids Phenylalanine and Tyrosine), 1199.54 (C-O COOH), 1135.67 (C-O), 600-800 (out of plane bending vibration C-H in amino acids Phenylalanine and Tyrosine); LC-MS (ESI) m/z Calcd for (19b) 629.29, Found m/z = 627.9 (M-1), m/z = 630.7 (M+1).
3-2- The Cyclic Longicalycinin A analogues
Synthesis of [Cyclo-(Gly-Tyr-Pro-Phe-Val)] (1)
Yield: 75%; White solid; IR (KBr): (cm-1) 3407 (NH amide), 1679.88 (C=O amide), 1622.02 (C=C in amino acids Phenylalanine and Tyrosine), 600-800 (out of plane bending vibration C-H in amino acids Phenylalanine and Tyrosine); 1HNMR (DMSO-d6, 300 MHz,): δ= 7.34-7.99 (m, CH-Ar), 8.11-8.27 (d, 1H, NHCO), 8.28-8.42 (s, br, 1H, NHCO), 8.47-8.81 (s, br, 3H, NHCO), 14.32-14.56 (s, br, OH, Tyrosine), 13C NMR (DMSO-d6, 75 MHz,) = 12.36 (CH3), 16.64, 17.97 (CH2 Proline), 23.66 (CH2-N Proline), 25.62 (CH2-Ar Phenylalanine), 25.72 (CH2-Ar Tyrosine), 41.89 (CH(CH3)2), 44.97 (CH2C=O), 46.02 (CH Tyrosine), 46.92 (CH Phenylalanine), 47.77 (CH Valine), 53.61 (CH Proline), 108.76, 109.69, 119.06, 120.37 (CH-Ar), 124.47 (C-Ar Tyrosine), 126.36 (CH-Ar), 127.49 (C-Ar Phenylalanine), 130.48 (C-OH Tyrosine), 137.06, 138.43, 141.00, 142.43, 142.88 (C=O) ; LC-MS (ESI) m/z Calcd for (1) 563.36, Found m/z = 564.10000(M+1).
Synthesis of [Cyclo-(Leu-Tyr-Pro-Phe-Ala)] (2)
Yield: 77%; White solid; IR (KBr): (cm-1) 3407 (NH amide), 1679.88 (C=O amide), 1619.02 (C=C in amino acids Phenylalanine and Tyrosine), 600-800 (out of plane bending vibration C-H in amino acids Phenylalanine and Tyrosine); 1HNMR (DMSO-d6, 300 MHz,): δ= 6.69-6.79 (d, 2H, CH-Ar Tyrosine), 7.06-7.85 (m, CH-Ar), 7.90-8.06 (t, 1H, CH-Ar Phenylalanine), 8.14-8.27 (d, 1H, NHCO), 8.35-8.51 (s, br, 1H, NHCO), 8.56-8.83 (s, br, 2H, NHCO), 13C NMR (DMSO-d6, 75 MHz,) = 12.35, 16.63 (CH3), 17.98 (CH2 Proline), 23.64 (CH Leucine), 25.61 (CH2 Proline), 25.73 (CH2 Phenylalanine), 25.84 (CH2 Tyrosine), 25.94 (CH2 Leucine), 41.81 (CH2-N Proline), 44.85 (CH Alanine), 45.93 (CH Leucine), 46.87 (CH Tyrosine), 47.75 (CH Phenylalanine), 53.55 (CH-N Proline), 108.78, 119.07, 120.37, 126.35 (CH-Ar), 127.28 (C-Ar Tyrosine), 127.47 (CH-Ar), 130.46 (C-Phenylalanine), 142.41 (C-OH Tyrosine) ; LC-MS (ESI) m/z Calcd for (2) 591.39, Found m/z = 592.40000(M+1).
[Cyclo-(Gly-Tyr-Pro-Phe-Ala)] (3) Synthesis of
Yield: 78%; White solid; IR (KBr): IR (KBr): (cm-1) 3205.47 (NH amide), 1676.03 (C=O amide), 1610.80 (C=C in amino acids Phenylalanine and Tyrosine), 600-800 (out of plane bending vibration C-H in amino acids Phenylalanine and Tyrosine); 1HNMR (DMSO-d6, 300 MHz,): δ= 7.45-7.55 (d of d, 2H, CH-Ar Phenylalanine), 7.64-7.75 (t, 1H, CH-Ar Phenylalanine), 7.79-7.88 (d, 2H, CH-Ar Phenylalanine), 7.90-8 (t, 1H,NHCO), 8.03-8.16 (d, 1H, NHCO), 8.31-8.46 (s, br, 1H, NHCO), 8.47-8.55 (d, 1H, NHCO), 8.70-8.79 (d, 1H, NHCO), 13C NMR (DMSO-d6, 75 MHz,) = 12.39 (CH3), 16.69, 18.03 (CH2 Proline), 41.49 (CH2C=O), 41.64 (CH2-N Proline), 41.81 (CH Alanine), 53.57 (CH Tyrosine), 55.15 (CH Phenylalanine), 62.18 (CH-N Proline), 114.52, 115.77, 120.72, 127.14 (CH-Ar), 128.82 (C-Ar Tyrosine), 132.66 (CH-Ar), 133.16 (C-Ar Phenylalanine), 133.34 (C-OH Tyrosine), 134.66, 139.68, 141.71, 150.09, 151.10 (C=O) ; LC-MS (ESI) m/z Calcd for (3) 535.33, Found m/z = 536.00000(M+1).
Synthesis of [Cyclo-(Ala-Tyr-Pro-Phe-Gly)] (4)
Yield: 80%; White solid; IR (KBr): (cm-1) 3209.33 (NH amide), 1674.10 (C=O amide), 1616.24 (C=C in amino acids Phenylalanine and Tyrosine), 600-800 (out of plane bending vibration C-H in amino acids Phenylalanine and Tyrosine); 1HNMR (DMSO-d6, 300 MHz,): δ= 7.55-7.66 (t, 1H, CH-Ar Phenylalanine), 7.74-7.85 (t, 2H, CH-Ar Phenylalanine), 7.90-8.09 (d, 2H, CH-Ar), 8.13-8.36 (d, 3H, NHCO), 8.42-8.64 (s, br, 1H, NHCO), 13C NMR (DMSO-d6, 75 MHz,) = 12.43 (CH3), 16.64, 18.04 (CH2 Proline), 23.60 (CH2 Phenylalanine), 25.58 (CH2 Tyrosine), 25.73 (CH2-N Proline), 41.87 (CH2 Glycine), 44.97 (CH2-N Proline), 45.93 (CH Alanine), 47.72 (CH Tyrosine), 53.62 (CH-N Proline), 108.74, 120.36, 126.35, 127.48, (CH-Ar), 129.19 (C-Ar Tyrosine), 130.46 (CH-Ar), 134.11 (C-Ar Phenylalanine), 146.92 (C-OH Tyrosine), 150.11, 158.30, 164.65, 166.05, 168.36 (C=O) ; LC-MS (ESI) m/z Calcd for (4) 535.33, Found m/z = 536.00000(M+1).
Synthesis of [Cyclo-(Phe-Ser-Pro-Phe-Gly)] (5)
Yield: 80%; White solid; IR (KBr): (cm-1) 3427.27 (NH amide), 1674.10 (C=O amide), 1641.32 (C=C in amino acids Phenylalanine), 600-800 (out of plane bending vibration C-H in amino acids Phenylalanine); 1HNMR (DMSO-d6, 300 MHz,): δ= 4.04-4.10 (s, br, 2H, NHCH2CO), 4.29-4.38 (s, br, 1H, CH-N Proline), 6.71-8.14 (m, CH-Ar), 8.13-8.45 (s, br, 4H, NHCO) ; LC-MS (ESI) m/z Calcd for (5) 535.33, Found m/z = 536.20000(M+1).
Synthesis of [Cyclo-(Val-Tyr-Pro-Ile-Ala)] (6)
Yield: 74%; White solid; IR (KBr): (cm-1) 3209.33 (NH amide), 1676.03 (C=O amide), 1639.38 (C=C in amino acid Tyrosine), 600-800 (out of plane bending vibration C-H in amino acid Tyrosine); 1HNMR (DMSO-d6, 300 MHz,): δ= 1.60-2.01 (d, 3H, CH-CH3), 8.12-8.87 (s, br, 4H, NHCO); LC-MS (ESI) m/z Calcd for (6) 543.39, Found m/z = 544.70000(M+1).
Synthesis of [Cyclo-(Pro-Tyr-Val-Phe-Ala)] (7)
Yield: 77%; White solid; IR (KBr): (cm-1) 3205.47 (NH amide), 1676.03 (C=O amide), 1546.80 (C=C in amino acids Phenylalanine and Tyrosine), 600-800 (out of plane bending vibration C-H in amino acids Phenylalanine and Tyrosine); 1HNMR (DMSO-d6, 300 MHz,): δ= 4.05-4.09 (s, br, 1H, CH-N Proline), 4.31-4.37 (s, be, 1H, CH-N Proline), 6.70-6.80 (d, 2H, CH-Ar Tyrosine), 7.10-7.99 (m, CH-Ar), 8.15-8.57 (s, br, 4H, NHCO), 8.91-8.99 (s, br, 1H, OH Tyrosine) ; LC-MS (ESI) m/z Calcd for (7) 577.38, Found m/z = 578.10000(M+1).
Synthesis of [Cyclo-(Phe-Tyr-Pro-Val-Gly)] (8)
Yield: 79%; White solid; IR (KBr): (cm-1) 3444.53 (NH amide), 1679.88 (C=O amide), 1554.52 (C=C in amino acids Phenylalanine and Tyrosine), 600-800 (out of plane bending vibration C-H in amino acids Phenylalanine and Tyrosine); 1HNMR (DMSO-d6, 300MHz,): δ= 7.46-7.52 (d of d, 2H, CH-Ar Phenylalanine), 7.68-7.72 (d, 2H, CH-Ar), 7.93-7.98 (d, 2H, CH-Ar), 8.47-8.50 (d, 1H, NHCO), 8.50-8.53 (d, 1H, NHCO), 8.72-8.74 (d, 1H, NHCO), 8.74-8.75 (d, 1H, NHCO), 13C NMR (DMSO-d6,75 MHz,) = 12.29 (CH3), 16.66, 17.98 (CH2 Proline), 18.68 (CH Valine), 26.81 (CH2 Glycine), 40.65 (CH2-N Proline), 41.75 (CH Tyrosine), 49.09 (CH Phenylalanine), 53.49 (CH-NH Valine), 78.64 (CH-N Proline), 119.07, 120.67, 128.69, 132.15 (CH-Ar), 132.24 (C-Ar Tyrosine), 133.10 (CH-Ar), 134.09 (C- Phenylalanine), 134.65 (C-OH Tyrosine), 139.69, 151.03, 151.08, 151.13, 158.66 (C=O) ; LC-MS (ESI) m/z Calcd for (8) 563.36, Found m/z = 564.30000(M+1).
Synthesis of [Cyclo-(Phe-Tyr-Pro-Phe-Val)] (9)
Yield: 75%; White solid; IR (KBr): (cm-1) 3444.57 (NH amide), 1674.10 (C=O amide), 1600.81 (C=C in amino acids Phenylalanine and Tyrosine), 600-800 (out of plane bending vibration C-H in amino acids Phenylalanine and Tyrosine); 1HNMR (DMSO-d6, 300 MHz,): δ= 4.39-4.41 (s, br, CH-N Proline), 6.73-6.76 (d, 2H, CH-Ar Tyrosine), 7.11-7.15 (d, 2H, CH-Ar, Tyrosine), 7.47-7.54 (d of d, 4H, CH-Ar, Phenylalanine), 7.66-7.74 (t, 1H, CH-Ar Phenylalanine), 7.80-7.86 (d, 2H, CH-Ar Phenylalanine), 7.91-7.99 (t, 1H, CH-Ar Phenylalanine), 8.05-8.15 (d, 2H, CH-Ar Phenylalanine), 8.49- 8.52 (d, 1H, NHCO), 8.52-8.55 (d, 1H, NHCO), 8.73-8.75 (d, 1H, NHCO), 8.76-8.78 (d, 1H, NHCO), 13.61-13.98 (s, br, 1H, OH Tyrosine), 13C NMR (DMSO-d6, 75 MHz,) = 10.86 (CH3), 16.69, 18.06 (CH2 Proline), 26.42 (CH Valine), 33.5 (CH2 Phenylalanine), 38.27 (CH2 Tyrosine), 41.48 (CH2-N Proline), 41.76 (CH Tyrosine), 45.02 (CH Phenylalanine), 46.63 (CH Phenylalanine), 56.85 (CH Valine), 68.10 (CH Proline), 114.48, 120.73, 127.11, 128.86 (CH-Ar), 132.65 (C-Ar Tyrosine), 133.11 (CH-Ar), 134.63 (C-Phenylalanine), 139.62 (C-OH Tyrosine), 149.50, 150.02, 151.14 (C=O); LC-MS (ESI) m/z Calcd for (9) 653.41, Found m/z = 654.20000(M+1).
Synthesis of [Cyclo-(Phe-Tyr-Pro-Phe-Ala)] (10)
Yield: 75%; White solid; IR (KBr): (cm-1) 3379.05 (NH amide), 1602.74 (C=O amide), 1558.38 (C=C in amino acids Phenylalanine and Tyrosine), 600-800 (out of plane bending vibration C-H in amino acids Phenylalanine and Tyrosine); 1HNMR (DMSO-d6, 300 MHz,): δ= 7.47-7.53 (m, CH-Ar), 8.50-8.52 (d, 1H, NHCO), 8.52-8.55 (d, 1H, NHCO), 8.73-8.75 (d, 1H, NHCO), 8.75-8.77 (d, 1H, NHCO), 13.72-13.89 (s, br, 1H, OH Tyrosine) ; LC-MS (ESI) m/z Calcd for (10) 625.38, Found m/z = 626.10000(M+1).
Synthesis of [Cyclo-(Thr-Val-Pro-Phe-Ala)] (11)
Yield: 76%; White solid; IR (KBr): (cm-1) 3211.26 (NH amide), 1676.03 (C=O amide), 1598.88 (C=C in amino acid Phenylalanine), 600-800 (out of plane bending vibration C-H in amino acid Phenylalanine); 1HNMR (DMSO-d6, 300 MHz,): δ= 3.50-3.70 (s, br, 1H, OH), 7.42-7.55 (d of d, CH-Ar), 7.67-7.73 (d, CH-Ar), 7.93-7.99 (d, CH-Ar), 8.26-8.43 (d, 2H, NHCO), 8.45-8.56 (d, 1H, NHCO), 8.67-8.82 (d, 1H, NHCO), 13C NMR (DMSO-d6,75 MHz,) = 12.44 (CH3), 16.70, 18.05 (CH2-N Proline), 41.88 (CH Valine), 53.61 (CH Alanine), 53.65 (CH Phenylalanine), 120.76, 128.87, 134.71 (CH-Ar), 139.71 (C-Ar Phenylalanine), 151.04, 151.16, 151.27 (C=O) ; LC-MS (ESI) m/z Calcd for (11) 515.36, Found m/z = 516.20000(M+1).
Synthesis of [Cyclo-(Phe-Tyr-Pro-Phe-Ile)] (12)
Yield: 78%; White solid; IR (KBr): (cm-1) 3396.41 (NH amide), 1672.17 (C=O amide), 1600.81 (C=C in amino acids Phenylalanine), 600-800 (out of plane bending vibration C-H in amino acids Phenylalanine); 1HNMR (DMSO-d6, 300 MHz,): δ= 3-3.04 (m, 6H, CH2-Ph), 6.69-6.78 (m, 4H, CH-Ar Tyrosine), 7.10- 7.17 (t, 2H, CH-Ar Phenylalanine), 7.47-7.50 (d, 1H, NHCO), 7.50-7.53 (d, 1H, NHCO), 7.69- 7.73 (d, 2H, CH-Ar), 7.81-7.85 (d, 2H, CH-Ar), 7.94-7.99 (d, 2H, CH-Ar), 8.08-8.12 (d, 2H, CH-Ar), 8.49-8.51 (d, 1H, NHCO), 8.52-8.54 (d, 1H, NHCO), 13C NMR (DMSO-d6, 75 MHz,) = 12.41, 16.70 (CH3), 18.05 (CH2 Isoleucine), 38.22 (CH2 Phenylalanine), 41.50 (CH2 Tyrosine), 41.81 (CH2-N Proline), 53.53 (CH Tyrosine), 53.62 (CH Phenylalanine), 118.78, 120.70, 128.86, 129.35 (CH-Ar), 132.65 (C-Ar Tyrosine), 133.31 (CH-Ar), 134.63 (C-Ar Phenylalanine), 139.64 (C-OH Tyrosine), 151.06, 151.16, 157.31, 158.09, 158.57 (C=O) ; LC-MS (ESI) m/z Calcd for (12) 667.42, Found m/z = 668.60000(M+1).
Synthesis of [Cyclo-(Val-Thr-Pro-Phe-Leu)] (13)
Yield: 80%; White solid; IR (KBr): (cm-1) 3375.20 (NH amide), 1674.10 (C=O amide), 1598.88 (C=C in amino acid Phenylalanine), 600-800 (out of plane bending vibration C-H in amino acid Phenylalanine); 1HNMR (DMSO-d6, 300 MHz,): δ= 3.98-4.05 (s, 1H, CH2NHCO), 4.07-4.11 (d, 1H, CH2NHCO), 4.31-4.43 (m, 1H, CH2-N Proline), 7.09-7.18 (t, 2H, CH-CH2-Ph), 7.46-7.53 (d of d, CH-Ar), 7.65-7.73 (t, CH-Ar), 7.80-7.86 (d, CH-Ar), 8.06-8.12 (d, 1H, NHCO), 8.24-8.37 (s, br, 1H, NHCO), 8.47- 8.55 (d, 1H, NHCO), 8.71-8.78 (d, 1H, NHCO), 13C NMR (DMSO-d6, 75 MHz,) = 12.47 (CH3), 16.70, 18.07 (CH2 Proline), 41.85 (CH2 Leucine), 53.63 (CH Leucine), 120.74, 128.86, 134.70 (CH-Ar), 139.68 (C-Ar), 151.11 (C=O); LC-MS (ESI) m/z Calcd for (13) 577.41, Found m/z = 578.20000(M+1).
Synthesis of [Cyclo-(Pro-Tyr-Phe-Phe-Leu)] (14)
Yield: 72%; White solid; IR (KBr): (cm-1) 3207.40 (NH amide), 1666.38 (C=O amide), 1598.88 (C=C in amino acids Phenylalanine and Tyrosine), 600-800 (out of plane bending vibration C-H in amino acids Phenylalanine and Tyrosine); 1HNMR (DMSO-d6, 300 MHz,): δ= 3.99-4.10 (m, 1H, CH-N Proline), 4.32-4.42 (m, 1H, CH-N Proline), 4.65-4.71 (s, br, 1H, OH Tyrosine), 7.06-7.29 (m, CH-Ar), 7.44-7.57 (d of d, 2H, CH-Ar), 8.22-8.43 (d, 2H, NHCO), 8.46-8.58 (d, 1H, NHCO), 8.70-8.80 (d, 1H, NHCO), 13C NMR (DMSO-d6, 75 MHz,) = 12.44 (CH3), 16.70, 18.04 (CH2 Proline), 41.84 (CH2 Leucine), 53.60 (CH Leucine), 120.75, 128.86, 134.66 (CH-Ar), 139.66 (C-Ar), 151.14 (C=O); LC-MS (ESI) m/z Calcd for (14) 667.42, Found m/z = 668.40000(M+1).
Synthesis of [Cyclo-(Pro-Tyr-Gly-Phe-Val)] (15)
Yield: 73%; White solid; IR (KBr): (cm-1) 3420 (NH amide), 1676.03 (C=O amide), 1537.16 (C=C in amino acids Phenylalanine and Tyrosine), 600-800 (out of plane bending vibration C-H in amino acids Phenylalanine and Tyrosine); 1HNMR (DMSO-d6, 300 MHz,): δ= 3.99-4.05 (s, 1H, CH2NHCO), 4.06-4.11 (d, 1H, CH2NHCO), 4.32-4.42 (m, 1H, CH2-N Proline), 6.70-6.79 (d, 2H, CH-Ar Tyrosine), 7.09-7.19 (t, 2H, CH-CH2-Ph), 7.45-7.55 (d of d, CH-Ar), 7.63-7.75 (t, CH-Ar), 7.77-7.88 (d, CH-Ar), 7.90-8 (t, CH-Ar), 8.05-8.14 (d, 1H, NHCO), 8.26-8.46 (s, br, 1H, OH Tyrosine), 8.47- 8.54 (d, 1H, NHCO), 8.56-8.63 (d, 1H, NHCO), 8.71-8.78 (d, 1H, NHCO), 8.86-8.93 (d, 1H, NHCO), 13C NMR (DMSO-d6, 75 MHz,) = 12.39 (CH3), 16.69, 18.09 (CH2 Proline), 38.25 (CH2 Phenylalanine or CH2 Tyrosine), 41.54 (CH2 Glycine), 41.88 (CH2-N Proline), 53.63 (CH Phenylalanine or CH Tyrosine), 115.80, 118.85, 127.21, 129.44 (CH-Ar), 132.68 (C-Ar Tyrosine), 133.21 (CH-Ar), 133.40 (C-Ar Phenylalanine), 134.71 (C-OH Tyrosine), 150.12, 151.19, 157.42, 158.34, 158.83 (C=O) ; LC-MS (ESI) m/z Calcd for (15) 563.36, Found m/z = 564.50000(M+1).
Synthesis of [Cyclo-(Phe-Thr-Pro-Phe-Val)] (16)
Yield: 76%; White solid; IR (KBr): (cm-1) 3439.34 (NH amide), 1674.10 (C=O amide), 1600.81 (C=C in amino acids Phenylalanine), 600-800 (out of plane bending vibration C-H in amino acids Phenylalanine); 1HNMR (DMSO-d6, 300 MHz,): δ= 6.70-6.78 (m, 2H, CH-Ar), 7.10-7.26 (m, 4H, CH-Ar), 7.26-7.56 (d of d, 4H, CH-Ar), 7.64-7.76 (t, 1H, CH-Ar), 7.78-7.89 (d, 4H, CH-Ar), 7.90-8.01 (t, 1H, CH-Ar), 8.05-8.16 (d, 1H, NHCO), 8.21-8.34 (s, br, 1H, OH Threonine), 8.38-8.44 (d, 1H, CH2-NHCO), 8.46-8.56 (d, 1H, NHCO), 8.71-8.80 (d, 1H, NHCO), 8.82-8.87 (d, 1H, CH2NHCO), 13C NMR (DMSO-d6, 75 MHz,) = 12.46, 16.71 (CH2 Proline), 18.06 (CH2 Phenylalanine), 41.50 (CH2 Glycine), 41.76 (CH2-N Proline), 41.86 (CH Threonine), 53.63 (CH Phenylalanine), 114.53, 127.17, 132.67 (CH-Ar), 133.19 (C-Ar Phenylalanine), 134.67, 139.66, 150.09, 151.16 (C=O) ; LC-MS (ESI) m/z Calcd for (16) 591.39, Found m/z = 592.40000(M+1).
Synthesis of [Cyclo-(Phe-Ser-Pro-Phe-Ala)] (17)
Yield: 77%; White solid; IR (KBr): (cm-1) 3203.54 (NH amide), 1676.03 (C=O amide), 1575.73 (C=C in amino acids Phenylalanine), 600-800 (out of plane bending vibration C-H in amino acids Phenylalanine); 1HNMR (DMSO-d6, 300 MHz,): δ= 7.06-7.32 (m, 2H, CH-Ar), 7.40-7.44 (d, 2H, CH-Ar), 7.35-7.57 (d, 2H, CH-Ar), 7.69-7.73 (d, 2H, CH-Ar), 7.94-7.99 (d, 2H, CH-Ar), 8.13-8.42 (s, br, 4H, NHCO) ; LC-MS (ESI) m/z Calcd for (17) 549.34, Found m/z = 550.30000(M+1).
Synthesis of [Cyclo-(Phe-Ser-Pro-Ala-Gly)] (18)
Yield: 76%; White solid; IR (KBr): (cm-1) 3454.34 (NH amide), 1676.03 (C=O amide), 1633.59 (C=C in amino acid Phenylalanine), 600-800 (out of plane bending vibration C-H in amino acid Phenylalanine); 1HNMR (DMSO-d6, 300 MHz,): δ= 7.11-7.28 (d, CH-Ar), 8.36-8.74 (s, br, 4H, NHCO) ; LC-MS (ESI) m/z Calcd for (18) 459.3, Found m/z = 460.20001(M+1).
Synthesis of [Cyclo-(Phe-Tyr-Pro-Phe-Gly)] (Cyclic Longicalycinin A) (19)
Yield: 78%; White solid; IR (KBr): (cm-1) 3420.12 (NH amide), 1679.88 (C=O amide), 1610.95 (C=C in amino acids Phenylalanine and Tyrosine), 600-800 (out of plane bending vibration C-H in amino acids Phenylalanine and Tyrosine); 1HNMR (DMSO-d6, 300 MHz,): δ= 3.09-3.14 (d of d, 2H, CH2-Ph), 8.22-8.31 (s, br, 4H, NHCO), 13C NMR (DMSO-d6, 75 MHz,) = 25.86 (CH3), 25.97, 26.98 (CH2 Proline), 27.15 (CH2 Phenylalanine), 41.80 (CH2-N Proline), 44.75, 45.91, 45.96, 47.74 (CH2), 53.52 (CH-N Proline), 124.42, 128.05, 129.14 (CH-Ar), 130.5 (C-Ar Phenylalanine), 158.39, 158.53, 158.81, 168.67, 169.03 (C=O); LC-MS (ESI) m/z Calcd for (19) 611.36, Found m/z = 612.50000(M+1).
MTT assay results
The deprotected linear and cyclic peptides of Longicalycinin A analogues have shown cytotoxicity to HepG2 and HT-29 cancer cell lines in varying degree (ranging IC50 values from 8.76 to 17.2µg/mL and 9.06 to 17.34µg/mL, respectively). Tables 1 and 2 show the IC50 results for all deprotected linear and cyclic analogues of Longicalycinin A peptide, respectively, along with IC50 results for 5-flurouracil, chosen as a standard toxic drug. Tables 3 and 4 contain viability percentage data for linear and cyclic analogues of Longicalycinin A peptide, respectively, on three cell lines; skin Fibroblast, HepG2 and HT-29 cancer cells.
Table 1. IC50 values (μg/mL) for anti-proliferative activity of peptides on HepG2 and HT-29 cells in MTT assay. Values are presented as mean ± SD of three independent experiments (n= 3). The stars show the values are significantly different from the corresponding control (*P <0.05, **P <0.01, *** P <0.001).
Table 2. IC50 values (μg/mL) for toxicity activity of peptides on HepG2 and HT-29 cells in MTT assay. Values are presented as mean ± SD of three independent experiments (n= 3). The stars show the values are significantly different from the corresponding control (*** P <0.001).
LDH analysis results
The results of viability percentage peptides-treated HepG2 and HT-29 cells calculated by measuring optical density in the LDH assay are shown in Tables 5 and 6.
Table 5. LDH assay of linear Longicalycinin A analogues for cell lines HepG2 and HT-29
Table 6. LDH assay of cyclic Longicalycinin A analogues for cell lines HepG2 and HT-29.
Flow cytometry analysis results
Tables 7 and 8 show flow cytometery results of linear and cyclic analogues of Longicalycinin A. Figures 1 and 2 show the histograms 1 and 5 as example. All the other related histograms are included in the supplementary file. The left dot plots present the forward scatter (FSC) parameter in the horizontal axis and side scatter (SSC) parameter in the vertical axis, which can be correlated with the relative size and granularity of the cells, respectively. The histograms show the intensity of fluorescence of the samples in the FL-2 channel, which corresponds to PI emission wavelength.26
Table 7. Apoptosis percentageof linear Longicalycinin A analogues for cell lines HepG2 and HT-29
Table 8. Apoptosis percentageof cyclic Longicalycinin A analogues for cell lines HepG2 and HT-29
Figure 1. Flow cytometery results obtained from deprotected linear peptide-treated HepG2 and HT-29 cells
Figure 2. Flow cytometery results obtained from deprotected cyclic peptide-treated HepG2 and HT-29 cells
Determination of Lysosomal membrane integrity results
Lysosome damage using acridine orange as a probe was assayed in isolated lysosome obtained from the treated HepG2 and HT-29. Values are presented as mean ± SD of three independent experiments performed for determination of Lysosomal membrane integrity of HepG2 and HT-29 cells (Figs 3-6). As shown in Figs 3 and 5, linear peptides with numbers 18 and 17 caused the highest absorption of acridine orange dye in HepG2 and HT-29 cell lines, respectively and in Figs 4 and 6, cyclic peptides with numbers 14 and 11 caused the highest absorption of acridine orange dye in HepG2 and HT-29 cell lines, respectively.
Figure 3. Determination of Lysosomal membrane integrity assay on HepG2. The values show acridine orange absorbance of the damaged lysosomes of peptide-treated HepG2 cells and are presented as mean ± SD of three independent experiments (n= 3). The stars show the values are significantly different from the corresponding control (**P <0.01, *** P <0.001).
Figure 4. Determination of Lysosomal membrane integrity assay on HepG2. The values show acridine orange absorbance of the damaged lysosomes of peptide-treated HepG2 cells and are presented as mean ± SD of three independent experiments (n= 3). The stars show the values are significantly different from the corresponding control (*P <0.05, **P <0.01, *** P <0.001).
Figure 5. Determination of Lysosomal membrane integrity assay on HT-29. The values show acridine orange absorbance of the damaged lysosomes of peptide-treated HT-29 cells and are presented as mean ± SD of three independent experiments (n= 3). The stars show the values are significantly different from the corresponding control (*P <0.05, **P <0. 01, *** P <0.001).
Figure 6. Determination of Lysosomal membrane integrity assay on HT-29. The values show acridine orange absorbance of the damaged lysosomes of peptide-treated HT-29 cells and are presented as mean ± SD of three independent experiments (n= 3). The stars show the values are significantly different from the corresponding control (*P <0.05 and *** P <0.001).
Discussion:
Two-step synthesis of peptides on 2-CTC resin was chosen as the main strategy in this study due to several advantages achievable from this kind of resin. At the end of synthesis to obtain protected linear peptides, the trityl linker could be readily cleaved under a mild acidic condition by treatment with TFA 1% in dichloromethane owing to the high stability of trityl cations attached to the resin.28 Meanwhile desired Longicalycinin A analogues with protected side chains could be obtained in a good yield and high purity without producing byproducts due to side reactions of the protecting groups released in the solution. In the second step, deprotection of the side chain (O-t-Bu) linear Longicalycinin A analogues were achieved with TFA 95% containing scavengers. For the synthesis of cyclic peptides, cyclization of linear (Ot-Bu) Longicalycinin A analogues was achieved using coupling reagents and then the final deprotection was applied on the cyclic (Ot-Bu) Longicalycinin A analogues by treatment with TFA 95% containing scavengers to obtain cyclic Longicalycinin A analogues.
Synthesized linear and cyclic Longicalycinin A analogues exhibited considerable cytotoxic activity against cell lines of HepG2 (human liver cancer Cell Line) and HT-29 (Human Colorectal Adenocarcinoma Cell Line) with mean IC50 values ranging from 8.76 to 17.2 µg/mL and 9.06 to 17.34µg/mL, respectively, in comparison with standard drug 5-fluorouracil (5-FU). The results of MTT assay are shown in Table 1. According to these results, the synthesized compounds 2, 3, 5, 7, 9, 10, 11, 15 and 19 with IC50 values (8.76-10.5 µg/mL) on HepG2 cell lines showed relatively high toxic activity against cell viability. All of the compounds had high toxic effects on colon cancer cell line with the exception of compounds 9 and 15 (see Table 3). Compounds 1, 8, 12, 13, 14, 16 and 18 with IC50 values (9.26-11.32 µg/mL) on HepG2, compounds 9 and 15 with IC50 values (10.44-10.55 µg/mL) on HT-29 cell lines showed low potency on cell death and were classified as fewer toxic agents on cell lines, assuming at least containing less than one third potential activity as that of 5-FU. Skin Fibroblast cells as the normal cells with a relatively high-rate division could provide a safety profile for the synthesized peptide compounds. The higher IC50 should give the better safety profile. Among the compounds thus prepared, compounds 1, 6, 8, 12, 13, 14 and 18 showed a high safety result on normal cell line with a low toxicity activity on HepG2 cell line. Moreover, comparing with linear Longicalycinin A, compounds 1, 8, 12, 13 and 14 give higher toxicity on cancerous HT-29 cell line. Structurally, compound 1 is different from Longicalycinin A linear peptide by two amino acids Gly and Phe being replaced by Val and Gly, respectively. Compound 8 is different from Longicalycinin A linear peptide by one amino acid Phe being replaced by Val. Compound 12 is different from Longicalycinin A linear peptide by one amino acid Gly being replaced by Ile. Compound 13 is different from Longicalycinin A linear peptide by three amino acids Gly, Tyr and Phe being replaced by Leu, Thr and Val, respectively. Compound 14 is different from Longicalycinin A linear peptide by three amino acids Gly, Pro and Phe being replaced by Leu, Phe and Pro, respectively. Comparing substitution of amino acids as above can direct this notion that the lipophilicity of the peptide compounds is in favour of high toxicity towards cancerous colon cells (HT-29) along with low toxicity on Fibroblast cells.
The results of MTT assay for Longicalycinin A cyclic analogues are shown in Table 4. According to these results, the synthesized compounds 1, 2, 4, 9, 10, 11, 12, 14, 17 and 19 with IC50 values (9.4-12.37 µg/mL) on HepG2 and compounds 1, 2, 5, 9, 10, 11 and 17 with IC50 values (9.98-11.56 µg/mL) on HT-29 cell lines showed relatively high toxic activity against cell viability. Cyclic analogues in HepG2 cell line than HT-29 cell line have high toxic effects. Compound 8 with IC50 value (9.12 µg/mL) on HepG2, compounds 3, 6, 7, 8, 13, 15, 16 and 18 with IC50 values (9.06-12.67 µg/mL) on HT-29 cell lines showed low potency on cell death and were classified as fewer toxic agents on cell lines, assuming at least cont
