14-Residue peptaibol velutibol A from Trichoderma velutinum: its structural and cytotoxic evaluation

Velutibol A (1), a new 14-residue peptaibol was isolated from the Himalayan cold habitat fungus Trichoderma velutinum. The structural characterization was carried out by 1D and 2D NMR studies, and tandem mass studies, and Marfey's method aided in determining the stereochemistry of the amino acids. The CD analysis revealed folding of the peptide in a 310-helical conformation. The intramolecular H-bonding was determined by an NMR-VT experiment. Cytotoxic evaluation was carried out against a panel of cancer cell lines. The cell cycle assay was carried out on human myeloid leukaemia (HL-60) cells and revealed the formation of apoptotic bodies and DNA damage in a dose-dependent manner. Three other peptaibols namely velutibol B (2), velutibol C (3), and velutibol D (4) were also isolated in trace amounts from the psychotropic fungus and characterized through tandem mass spectroscopy and Marfey's analysis.

In our previous report, four lipopeptaibols namely lipovelutibols A-D from fungus T. velutinum were isolated from soils of Himalayan cold habitat. 33 In continuing the exploration of peptaibiotics from T. velutinum, we herein, report isolation, structure elucidation, conformational analysis along with

Results and discussion
The chromatographic purication of the ethyl acetate extract was carried out through RP-HPLC, which gave 15 fractions (Fig. S1, ESI †). The re-purication of fraction-6, 7, 8 and 10 gave four lipopeptaibols as described previously. 33 The Fr-6 and Fr-7 also contained mixtures of peptides with higher m/z values, which were further puried through RP-HPLC to get the peptaibols (1)(2)(3)(4)  The detailed examination of 1D along with 2D experiments such as COSY, TOCSY, NOESY and HMBC data elucidated the structure as 1, which comprises 13 amino acids viz. six a-aminoisobutyric acids (Aib), two leucines (Leu), one valine (Val), three prolines (Pro) and one glutamine (Gln) and an amino alcohol leucinol (Leuol) at the C-terminus. The 14-residue sequence was found to have acetylation at the N-terminus (Fig. 2). For instance, the 2D experiments such as COSY and TOCSY showed three Pro, two Leu, one Leuol, and one Val moieties in the molecule. The amino acid Gln was recognized by the presence of correlation between two NHs having broad singlets at d H 6.95 and 7.48 in COSY and TOCSY experiments along with the correlations obtained from a-H, b-H 2 , and g-H 2 . The presence of six Aib was revealed by HMBC correlations as shown in Fig. 2 (Fig. S9-S13 (Fig. 3). The MS/MS data in combination with the NOESY and HMBC correlations established the sequence of compound 1 as Ac-Aib-Gln-Leu-Aib-Pro-Val-Leu-Aib-Pro-Aib-Aib-Aib-Pro-Leucinol.
The advanced Marfey's method 49 was used for the determination of stereochemistry. Acid hydrolysis of the compound 1 was done and derivatized with 1-uoro-2,4-dinitrophenyl-5-Lalaninamide ( L FDAA). Likewise, both the D-and L-amino acid standards were derivatized with L FDAA. The amino acid glutamine gets converted into glutamic acid on acid hydrolysis, 35 thus L Glu and D Glu was derivatized for stereochemical determination of Gln. The amino acid standards thus obtained were compared with L-FDAA derivatives of acid hydrolyzed compounds using LCMS instrument. The retention times for standard amino acid-L FDAA derivatives were found to be L Glu/ D Glu  The probable primary sequence of these peptides has been given in Table 2    Conformational studies were performed through CD technique 50,51 and NMR-VT (variable temperature). 52,53 The CD spectra of the 1 in methanol were analyzed as shown in Fig. 4.
The shapes of the CD pattern showed a Àive maxima at 206 nm and a Àive shoulder at 220 nm (in the vicinity of 222 nm). The ellipticity ratio R [q T 220 ]/[q T 206 ] was found to be 0.23. This clearly indicated that the compound 1 folds in a 3 10 -helix in methanol as the value for R was less than 0.50, which is known for the high propensity of 3 10 -helix. The NMR-VT examined the manner in which the NH groups in the peptide behaved as a function of temperature. 52,53 1 H NMR of peptaibol 1 was recorded at four different temperatures i.e. 298 K, 308 K, 318 K, 328 K in DMSO-d6. Out of the thirteen NH protons in the putative peptide, the chemical shis of NH-1, NH-2, NH-5 along with amide NH 2 of Gln (NH-8 and NH-13) were sensitive to heating while the other NH groups had shown very little change in chemical shi values as shown in Fig. 5. On the basis of these 1 H NMR-VT experiment results (Fig. S35, ESI †), it was evident that NH-3 (Leu-3), NH-4 (Aib-4), NH-6 (Leu-7), NH-7 (Aib-8), NH-9 (Aib-10), NH-10 (Aib-11), NH-11 (Aib-12), and NH-12 (Leuol) were involved in intramolecular hydrogen bonding in peptaibol 1.   The cytotoxicity testing of compound 1 was performed against various cell lines, which includes human lung cancer (A549), human colon cancer (LS-180), human myeloid leukemia (HL-60) and human breast cancer (MDA-MB-231) using MTT (3-(4,5dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide). Herein, paclitaxel was used as positive control for cytotoxic assay. The MTT assay showed inhibitory activity of compound 1 against MDA-MD-231 and HL-60 cancer cell lines having IC 50 values of 7 mM and 4 mM respectively ( Table 3). The IC 50 for positive control was found to be in range of 2.0 to 6.8 nM against four cancer cell lines tested. Further, the cell-cycle assay was performed on HL-60 cell lines. The compound 1 was treated against HL-60 cells at concentrations 5, 10, 20 and 30 mM and incubated for 24 h. The compound 1 showed DNA damage in a dose-dependent manner. The HL-60 cells, with no treatment, showed non-signicant 2% apoptotic DNA whereas the cells treated with 1 showed 70%, 85% and 99% apoptotic DNA population at 10, 20 and 30 mM concentrations respectively (Fig. 6a). Apoptotic bodies are small DNA fragments wrapped around the single membrane vesicles mainly formed when cells undergo apoptosis. These apoptotic bodies can be visualized under a uorescent microscope by using DNA staining dyes such as 4 0 ,6-diamidino-2-phenylindole (DAPI). The HL-60 cells were incubated with the compound 1 at different concentrations (5, 10, 20 and 30 mM) for 24 h. The apoptotic bodies (indicated by white arrows in Fig. 6b) were seen in cells treated with tested compound whereas healthy and round nuclei with no DNA fragmentation was observed with the untreated cells. Formation of apoptotic bodies increased in a dosedependent manner. Similarly, the phase contrast microscopy of cells treated with 1 showed bled and distorted cells while the control cells were observed as healthy (Fig. 6c). The % apoptosis is also represented graphically in Fig. 7.
The compound 1 was also tested for anti-tubercular potential. The Minimum Inhibitory Concentration (MIC) of compound 1 was found to be 32 mg mL À1 against Mycobacterium tuberculosis. Rifampicin was used as a positive control with MIC 0.06 mg mL À1 (Fig. S36, ESI †).

Fungal cultivation
The isolation, microbial identication, characterization and cultivation for Trichoderma velutinum has been described in previous reports. 33,54 Isolation of compounds (1-4) The semi-preparative HPLC purication was performed using reverse-phase (RP) column (Reprosil Gold C 18 , 10 Â 250 mm, 5 mm, Dr Maisch GmbH) on ThermoFinnigan HPLC system. The purication details were same as provided in the previous report.33 The Fr-6 on further isolation gives three fractions (Fig. S2, ESI †), one was lipovelutibol A, the other two being velutibol A (1) and velutibol B (2) in 3.5 mg and 0.3 mg respectively. Similarly, Fr-7 gave two fractions, the rst being lipovelutibol B, and the second fraction (0.4 mg) was re-puried through-HPLC using RP column (LiChrospher RP18e, 125 Â 4 mm, 5 mm, Merck) on Shimadzu HPLC system through a gradient program comprising 0.1% TFA in water as mobile phase A and acetonitrile as mobile phase B. The method used was initiated with 10% B for 5 min then gradually increased to 60% B in next 30 min, kept at 60% B for 10 min, then gradually decreased to 10% B in 5 min and maintained at 10% B for next 10 min, with an overall run time of 60 min at 1 mL min À1 ow rate of and observed at 214 nm wavelength. This gave two fractions as velutibol C (3) (0.2 mg approx.) and velutibol D (4) (0.1 mg approx.) (Fig. S3, ESI †).

HPLC purity analysis
The HPLC analysis was carried out on Shimadzu HPLC system coupled with a PDA detector, thermostat and quaternary pump using a RP column (LiChrospher RP18e, 5 mm, 250 Â 4 mm, Merck). The mobile phase A comprises of 0.1% formic acid in water and mobile phase B as acetonitrile. The gradient program was started initially by keeping 10% B for 5 min, gradually increased from 10-60% B in next 30 min, remain at 60% B for next 10 min, followed by gradual decrease from 60-10% B in 5 min and nally remained at 10 for next 13 min with an overall run time of 63 min. It was detected at 214 nm with 1 mL min À1 ow rate.

LCHRMS analysis
The HRMS and tandem mass spectroscopy was carried out on Agilent HRMS 6450 4HD Q-TOF Mass Spectrometer coupled with LC system. The instrument was maintained at capillary voltage 3500 V, source temperature 350 C, 7.0 L min À1 gas ow and spray voltage of 4.5 KV with a resolution of 30 000 were used for HRMS analysis. The tandem mass spectroscopy (MS n ) was performed for the range of 100-2000 amu at fragmentor voltage at 135.0 V. 20 ions per cycle was selected for precursor ion with the minimum intensity of 5000 and collision induced dissociation (CID) at 30 and 40 V.
Amino acid conguration determination (Marfey's analysis). The Marfey's method was used for amino acid conguration determination. The compound 1 (approx 0.2 mg) is hydrolysed in 9 N HCl (0.5 mL) at 110 C for 18 h. The solution thus obtained was evaporated under vacuum to dryness. It was then dissolved in water (25 mL) and transferred to 2 mL vial. To this, 1 M solution of sodium bicarbonate (10 mL) and 1% solution of Marfey's reagent in acetone (50 mL) was added. The mixture thus obtained was heated at 40 C under agitation. Aer 1 h, 2 N HCl This journal is © The Royal Society of Chemistry 2020 RSC Adv., 2020, 10, 31233-31242 | 31239 (5 mL) was added to quench the reaction. It was dried under stream of air and reconstituted in methanol (0.2 mL) for further analysis. This was analysed by Shimadzu triple quad MS coupled with LC system using a RP column (LiChrospher RP18e, 5 mm, 250 Â 4 mm, Merck). The eluent A comprises of 0.1% formic acid in water and eluent B as acetonitrile was used through a gradient program of 10-60% B in 30 min, keeping 60% B for 10 min, reduced gradually to 10% B in 1 min and maintained at 10% B for next 4 min with 0.5 mL min À1 ow rate and overall runtime of 45 min. The amino acids (approx. 0.2 mg) was added with water (50 mL), 1 M sodium bicarbonate solution (20 mL) and 1% solution of Marfey's reagent in acetone (100 mL) in separate 2 mL vials and heated at 40 C. Aer 1 h, 2 N HCl (10 mL) was added to the reaction and was dried using air stream. Further, it was reconstituted in methanol (1 mL) and analysed using aforementioned method. This was then compared with LCMS chromatogram of derivatized compound 1 to establish amino acid conguration. The compound 2, 3 and 4 were analysed similarly on 20 mg scale to ascertain amino acid conguration. The amino acids L Ile and L allo-Ile were differentiated through a chiral LCMS. These were treated with Marfey's reagent as stated earlier and analysed using column (Merck, ChiraDex HR, 4.0 Â 250 mm) with gradient programme. The mobile phase consists of A (0.1% formic acid in water) and B (acetonitrile), with the gradient initiated at 5% B and gradually increased to 50% B in 35 min, stayed at 50% B for next 5 min followed by sharp decrease to 5% B in 2 min and stayed at the same for next 5 min making a overall run time of 47 min with a ow rate of 0.5 mL min À1 . The compounds 2, 3 and 4 previously treated with acid and derivatized with Marfey's reagent were compared with elution of amino acid standards.

NMR analysis
The 1D and 2D NMR experiments were recorded on a Bruker 400 MHz NMR instrument. The data was recorded in ppm scale using tetramethylsilane or residual NMR solvent resonance as a reference. All the 1D and 2D NMR spectra were processed via MestReNova. The NMR-VT experiment was carried out at Bruker 500 MHz spectrometer coupled with a thermostat. The data was acquired at four different temperatures i.e. at 298 K, 308 K, 318 K and 328 K.

CD analysis
The circular dichroism (CD) experiment was performed on a Jasco J-1500 CD spectrophotometer using 2 mm cuvette. The solution of compound 1 in methanol was prepared at 0.1 mg mL À1 concentration. The spectrum was recorded from 260 to 180 nm with 50 nm min À1 scanning speed at a response time of 1 s and 1 nm bandwidth. The data obtained was accumulation of two scans.

Cytotoxic assay (MTT-assay)
The cytotoxic effect of compound 1 on cancer cells was assayed by using MTT dye. The HL-60 cell lines were seeded for 4 h in round bottom 96 well plates. It was then treated with different concentrations of compound 1 dissolved in DMSO and incubated for 48 h. With 3 h earlier to experiment termination, the MTT dye (20 mL) from the stock solution of 2.5 mg mL À1 was added to each well. The MTT-formazan crystals thus formed were dissolved in DMSO (160 mL) and observed at 570 nm. The untreated control culture was treated with DMSO (<0.2%).

Cell cycle analysis
The cell cycle phase distribution analysis in HL-60 cells was carried out by propidium iodide (PI) staining. The HL-60 cells were treated with different concentration (5, 10, 20 and 30 mM) of 1. Aer 24 h, the cells were centrifuged and collected at 400 Â g and washed two times with PBS followed by xing with icecold ethanol (70%). It was incubated overnight at 4 C. Next day, cells were centrifuged at 4000 rpm, collected and washed with PBS followed by incubation with RNase (0.2 mg mL À1 ) for 90 min at 37 C. It was then stained with PI (10 mg mL À1 ) and kept for 30 min in dark. The analysis of cells were carried out through ow-cytometer (FACS Calibur, Becton Dickinson) using list mode on 10 000 events for FL2-A vs. FL2-W. The ModFit soware was used for the calculation of uorescence intensity of the sub-G1 cell fraction representing the apoptotic cell population.
Hoechst staining HL-60 cells were incubated with compound 1 in a 12 well plate. Aer 24 h, cells were centrifuged at 400 Â g, collected and washed two times with PBS. The cells were xed using 1 mL solution of cold acetic acid in methanol (1 : 3) and incubated overnight at 4 C. Further, the cells were centrifuged at 400 Â g, collected and re-suspended in xing solution (100 mL). Further, the cells were spread on a slide and dried for 4-6 h at room temperature. Slides were then stained with DAPI in PBS containing 0.05% Tween 20 (5 mg mL À1 ) and incubated for 30 min followed by washing with water and PBS. The slide covered by a coverslip was mounted with uid containing 50 mL glycerol and PBS (1 : 1) and sealed with white nail polish. Nuclei were observed using microscope (20Â) and photographed by Olympus IX70 microscope. The treated and untreated control cells were simply photographed using a microscope.

Anti-tubercular assay
The MIC of compound 1 was determined by the micro-broth dilution method against M. tuberculosis H 37 Rv. 55 The bacterial cells was grown in Middlebrook 7H9 broth containing glycerol (0.5%), Tween 80 (0.05%), and ADC (10%) under constant shaking for 10 to 15 days at 37 C in 5% CO 2 . A suspension of M. tuberculosis cells was prepared in normal saline solution containing Tween 80 (0.5%). The turbidity of the resulting suspension was attuned by 1 McFarland (McF) standard (z1.0 Â 10 7 CFU mL À1 ). The bacterial suspension was further diluted 10 folds with the medium. The compound 1 was serially diluted in Middlebrook 7H9 to 2-folds in 100 mL per well in 96-well U bottom microtitre plates. A 100 mL of the diluted suspension was added to each well resulting in a nal inoculum of 1.0 Â 10 6 CFU mL À1 in the well, and the compound 1 was nally in range from 0.5 mg mL À1 to 128 mg mL À1 . The plates were incubated at 37 C for seven days in 5% CO 2 . The Resazurin Microtiter Assay (REMA) method was used for evaluation of the results. Aer incubation, resazurin (15 mL, 0.04%) and Tween 80 (12.5 mL, 20%) was added in each well of the plate. The plates were incubated for 48 h and were read visually to ascertain the MIC from the minimum concentration of the compound showing no change in color.

Conclusions
Herein, we report isolation of four 14-residue peptaibols from a psychrotrophic fungus T. velutinum of the Himalayan cold habitat. The complete structural characterization of 1 was established by intensive 1D, 2D NMR, and tandem mass spectroscopic studies. Whereas, the probable primary sequence for compound 2-4 was established using MS/MS studies. The stereochemical determination by Marfey's analysis revealed Lform of the amino acids in these putative peptides. The conformational studies carried out using CD spectroscopy revealed that compound 1 folds in a 3 10 -helix in methanol. The variable temperature experiment performed through NMR showed that NH-3 (Leu-3), NH-4 (Aib-4), NH-6 (Leu-7), NH-7 (Aib-8), NH-9 (Aib-10), NH-10 (Aib-11), NH-11 (Aib-12) and NH-12 (Leuol) were involved in intramolecular hydrogen bonding in peptaibol 1. These experiments also gave an insight into its secondary structure and may correlate to the biological activities asserted by 1. The cytotoxic assay showed compound 1 having IC 50 values of 7 mM and 4 mM against MDA-MD-231 and HL-60 cancer cells respectively. Further, the cell cycle assay revealed DNA damage and formation of apoptotic bodies in a dose-dependent manner against HL-60 cancer cells. The compound 1 also showed weak MIC of 32 mg mL À1 against M. tuberculosis.

Conflicts of interest
There are no conicts to declare.