General methods: All reactions were carried out under nitrogen atmosphere. Solvents were dried as follows: THF, DMF, CHCl from an auto-column distillation system. All other solvents and reagents, including diisopropylamine (DIPEA), were purchased as their driest form from different sources and used directly. Flash column chromatography was performed using a CombiFlash EZ Prep system (RediSep® Silica Gold Columns) using mixtures of increasing polarity. Silica pads/plugs were run using fritted Buchner funnels and silica Gel (Mesh 60). C NMR and H NMR spectra were recorded on Bruker 500 MHz Avance III AV-500 NMR spectrometer and CDCl (Cambridge Isotopes) was treated with flame-dried KCO (solid). For NMR data all chemical shifts (δ) are quoted in parts per million (ppm) referenced to the appropriate residual solvent peak (CHCl, 7.26 and 77.16 ppm), with the abbreviations s, br s, d, t, q, and m denoting singlet, broad singlet, doublet, triplet, quartet and multiplet respectively. Coupling constants (J) are provided in Hertz (Hz) and analyzed by MestReNova software and confirmed by manual calculation. High resolution mass spectrometry and optical rotation measurements ([α]D) for maj-o (1) were recorded on a Waters UHPLC-Q-ToF (Xevo) and Jasco P-200 polarimeter, respectively.
Dipeptide 3: Benzyl Boc-N-methyl-L-valinate HCl salt was prepared from Boc-N-Me-L-valine (Sigma-Aldrich, catalog number 15538) using previously described experimental procedures to provide multigram quantities of Benzyl Boc-N-methyl-L-valinate HCl salt (98% yield over 2 steps, see Fig. S1). To a solution of O-acetyl-N-(tert-butoxycarbonyl)-L-threonine (1.07 g, 4.09 mmol), and HATU (4.09 g, 4.09 mmol) in CHCl (11.1 mL) at 0˚C, was added DIPEA (1.4 mL, 7.98 mmol), and stirred for 1 h at 0˚C. After the activation, benzyl Boc-N-methyl-L-valinate HCl salt (1.0 g, 3.89 mmol) was added, and the reaction was allowed to slowly reach room temperature and stirred overnight. The reaction mixture was then concentrated under reduced pressure, diluted with diethyl ether (150 mL), quenched with 2 M HCl (100 mL), and extracted with diethyl ether (3 × 75 mL). The combined organic layers were washed with 2N HCl (3 × 75 mL), brine, dried over MgSO, and concentrated. The crude residue was purified via silica column chromatography (0-35% EtOAc in hexanes) to give benzyl N-(O-acetyl-N-(tert-butoxycarbonyl)-L-threonyl)-N-methyl-L-valinate 3 as a pale yellow oil (1.13 g, 2.43 mmol, 63%). H-NMR Spectroscopic data was in accordance with previously reported data (Fig. S2).
Tripeptide 5: To a solution of dipeptide 3 (1.13 g, 2.43 mmol) in THF (2.4 mL) was added 4N HCl in dioxane (12.2 mL, 48.6 mmol) at 0˚C. The reaction was allowed to reach room temperature and stirred for 3 h. The mixture was concentrated under reduced pressure to yield the corresponding HCl salt (978 mg, 2.44 mmol, quant.). The resulting salt product was combined with N-Boc-N-methyl-O-methyl-L-tyrosine (794 mg, 2.57 mmol), DIPEA (1.34 ml, 7.70 mmol) and DMF (7.33 mL), then cooled to 0˚C for 10 min. COMU (1.099 g, 2.57 mmol) was then added portion wise, and the reaction was stirred for 3 h at 0˚C and then partitioned between ethyl acetate (150 mL) and 1 N HCl (150 mL). The aqueous layer was extracted with ethyl acetate (3 × 100 mL) and the combined organic layers were washed with 1 M NaHCO (3 X 75 mL), HO and brine, dried over MgSO, filtered, and concentrated under vacuum. The resulting residue was purified via silica column chromatography (0-30% ethyl acetate in dichloromethane), providing benzyl Boc-N-methyl-O-methyl-L-tyrosinyl-N-(O-acetyl-N-(tert-butoxycarbonyl)-L-threonyl)-N-methyl-L-valinate 5 (918 mg, 1.40 mmol, 55%) as a white foam. H-NMR Spectroscopic data was in accordance with previously reported data (Fig. S3).
Majusculamide-o, 1: To a solution of tripeptide 5 (120 mg, 0.183 mmol) in THF (0.320 mL) was added HCl 4N in dioxane (1.830 mL, 7.32 mmol) at 0 °C. The reaction was stirred for 4 h at room temperature then concentrated under reduced pressure to afford the corresponding HCl salt (108 mg, 0.183 mmol, quantitative) as white semi-solid foam. In a separate reaction vessel, octanoic acid (28.9 mg, 0.201 mmol) and HATU (78 mg, 0.201 mmol) were combined in DMF (521 µl) at room temperature and DIPEA (35.0 µl, 0.201 mmol) was added dropwise. After 1 h stirring at room temperature, the mixture was transferred to a separate reaction vessel containing the tripeptide salt (108 mg, 0.182 mmol). Then, DIPEA (31.9 µl, 0.182 mmol) was added and the reaction was stirred overnight at rt. The reaction mixture was diluted with diethyl ether (15 mL) and quenched with 1N HCl (10 mL). The aqueous layer was extracted with diethyl ether (3 × 15 mL) and the combined organic layers were washed with 2N HCl (3 × 10 mL), water, brine, dried over MgSO4, and filtered. The organic layer was concentrated under reduced pressure to afford (benzyl N-(O-acetyl-N-((S)-3-(4-methoxyphenyl)-2-(N-methyloctanamido)propanoyl)-L-threonyl)-N-methyl-L-valinate 6 (110 mg, 0.161 mmol, 88% yield) as a pale yellow foam which was taken directly to the next step without further purification. A solution of benzyl N-(O-acetyl-N-((S)-3-(4-methoxyphenyl)-2-(N-methyloctanamido)propanoyl)-L-threonyl)-N-methyl-L-valinate (110 mg, 0.161 mmol) in 10% Pd/C (25.8 mg, 0.024 mmol) in MeOH (1.125 mL) was stirred under a hydrogen atmosphere for 1 h. The reaction mixture was quantitatively transferred and filtered using MeOH through a plug of Celite Filter Aid and concentrated to afford the debenzylated product, N-(O-acetyl-N-((S)-3-(4-methoxyphenyl)-2-(N-methyloctanamido)propanoyl)-L-threonyl)-N-methyl-L-valine (94.5 mg, 0.160 mmol, 99% yield), as a pale yellow foam. To a solution of the debenzylated product (94.5 mg, 0.160 mmol), pyrrolyl proline 7 (39.2 mg, 0.160 mmol), and DIPEA (84 µl, 0.479 mmol) in DMF (532 µl) was added COMU (70.5 mg, 0.160 mmol) at 0 °C. The reaction was allowed to reach rt and stirred for 2 h, then diluted with EtOAc (50 mL) and quenched with 1 M HCl (35 mL). The aqueous layer was extracted with EtOAc (3 × 25 mL) and the combined organic layers were washed with 1 M HCl (3 × 10 mL), water, brine, dried over MgSO, filtered and concentrated under reduced pressure. The resulting residue was purified via silica column purification (0-35% EtOAc in DCM) to afford 1 (75 mg, 0.096 mmol, 60% yield), maj-o, as a white foamy solid. [α] = -58.1 (c = 0.9, CHCl); H NMR (500 MHz, CDCl, mixture of rotamers, ca 4:1) δ 7.27 (d, J = 1.9 Hz, 1H), 7.10 (d, J = 8.4 Hz, 2H, major rotamer), 7.04 (dd, J = 30.0, 8.3 Hz, 2H, minor rotamer), 6.91 (d, J = 8.8 Hz, 1H, major rotamer), 6.82 (d, J = 8.8 Hz, 1H, minor rotamer), 6.79 (d, J = 8.4 Hz, 2H, major rotamer), 6.71 (d, J = 8.6 Hz, 2H, minor rotamer), 6.08 (d, J = 6.0 Hz, 1H), 5.66 (d, J = 9.3 Hz, 1H), 5.36 (m, 1H), 5.24 (dd, J = 6.4, 3.6 Hz, 1H), 4.99 (d, J = 11.1 Hz, 1H), 4.95 (dd, J = 8.9, 3.4 Hz, 1H), 4.81 (br q, J = 6.7 Hz, 1H), 4.37 (br s, 1H), 3.86 (m, 1H), 3.80 (dd, J = 11.7, 4.4 Hz, 1H), 3.76 (s, 3H), 3.21 (dd, J = 14.7, 6.7 Hz, 1H), 3.14 (s, 3H, minor rotamer), 3.06 (s, 3H, major rotamer), 2.95 (dd, J = 13.6, 8.5 Hz, 2H), 2.93 (s, 3H, minor rotamer), 2.87 (s, 3H, major rotamer), 2.46 (ddd, J = 14.5, 10.1, 4.7 Hz, 1H), 2.23 (t, J = 7.4 Hz, 2H), 2.03 (m, 1H), 1.99 (s, 3H, major rotamer), 1.98 (s, 3H, minor rotamer), 1.70 - 1.58 (m, 2H), 1.52 (m, 2H), 1.46 (d, J = 6.7 Hz, 3H), 1.30 - 1.27 (m, 6H), 1.17 (d, J = 6.4 Hz, 3H), 1.09 (m, 1H), 0.98 (d, J = 6.5 Hz, 3H, major and minor rotamer overlap), 0.87 (t, J = 6.9 Hz, 3H), 0.81 (d, J = 6.6 Hz, 3H, minor rotamer), 0.79 (d, J = 6.6 Hz, 3H, major rotamer).C NMR (126 MHz, CDCl) δ 174.68, 174.43, 170.53, 169.97, 169.93, 169.89, 169.02, 158.41, 154.28, 129.91, 128.99, 125.45, 114.00, 71.94, 68.83, 59.34, 58.71, 58.24, 57.59, 57.05, 55.28, 52.20, 36.72, 33.82, 32.97, 31.77, 30.55, 29.38, 29.17, 27.28, 25.11, 22.72, 21.14, 18.96, 18.52, 17.49, 17.04, 14.17. HRMS (ESI) m/z: calcd for CHNONa (M + Na) 806.4311, measured: 806.4316.
OVCAR3 and OVCAR8 are NCI-60 cell lines obtained from the National Cancer Institute Division of Cancer Treatment and Diagnosis Tumor Repository (NCI DCTD; Frederick, MD). Together with H125 (obtained from Dr. Frederick A Valeriote, Henry Ford Health, Detroit, MI), these three cell lines were grown in Roswell Park Memorial Institute medium 1640 (RPMI; Gibco, Cat# 11,875-093) supplemented with 10% fetal bovine serum (FBS; Gibco, Cat# A52567-01) and 1% Pen-Strep (100 U/mL penicillin, 100 μg/mL streptomycin; Gibco, Cat# 15,140-122) in a humidified 37 °C incubator with 5% CO. U251N, HEPG2, and PANC1 (also obtained through Dr. Valeriote) were grown in Dulbecco's Modified Eagle Medium (DMEM; Gibco, Cat# 11,965-092) supplemented with 10% FBS, 1% Pen-Strep, and 1% L-Glutamine (2 mM; Gibco, Cat# A2916801). All cell lines were tested for mycoplasma using a MycoAlert Mycoplasma Detection Kit (Lonza, Cat# LT07-118).
All cell lines were plated in 96-well plates (Corning, Cat# 3598) with 5 × 10 cells per well and treated with a serial dilution of maj-o after 24 h. Maj-o was dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich, Cat# 472301), and the DMSO concentration between the samples was normalized. Specific drug concentrations were optimized to generate full killing curves and are indicated in either the Fig. or Fig. legends. Cellular proliferation was measured using a CellTiter-Glo Luminescent Cell Viability Assay (Promega, Cat# G7573) at 72 h, and luminescence values were measured at an integration time of 20 ms on a Biotek Synergy Neo Plate Reader (Agilent).
OVCAR3 cells were plated in 24-well plates (Corning, Cat# 3526) with 5 × 10 cells per well and treated with 0, 5, 10, 20, and 30 nM maj-o after 24 h. Concentrations were chosen as sub-, 1x-, 2x-, and 3x-IC with 24, 48, and 72 h timepoints, and the DMSO concentration between samples was equalized. The optimal concentration of the dyes was determined by titration and calculating the SI (200 nM ApoTracker, 1:400 ZombieNIR). Cells and culture media were collected and stained with ApoTracker Green (BioLegend, Cat# 427402) and Zombie NIR (BioLegend, Cat# 423105) to assess apoptosis at 24, 48, and 72 h timepoints. Cells were stained in a 96-well rb plate (TFS, Cat# 163320), stabilized using 1% FBS in PBS, and fixed using 1% paraformaldehyde (PFA; EMS, Cat# 15710) in PBS. Samples were acquired on an Attune CytPix Flow Cytometer (ThermoFisher Scientific) located in the MSU Flow Cytometry Core Facility (supported by the Equipment Grants Program, award #2022-70410-38419, from the U.S. Department of Agriculture [USDA], National Institute of Food and Agriculture [NIFA]). Data was analyzed with FCS Express (DeNovo Software, version 7.24.0030), and FMOs were used as gating controls. The percentages of live (ApoTracker-/Zombie NIR-), early apoptotic (ApoTracker + /Zombie NIR-), and late apoptotic (ApoTracker + /Zombie NIR +) singlets were evaluated.
OVCAR3 cells were plated in 6-well plates (avantor, Cat# 10,062-892) with 5 × 10 cells per well and treated with 638 nM (500 ng/mL) maj-o after 24 h. Durations of treatment were either 0 h, 4 h, or 24 h, and the DMSO concentration between samples was normalized. The RNA was isolated and purified using a RNeasy Mini Kit (Qiagen, Cat# 74,104). RNA amounts were quantified with a DS-11 Spectrophotometer (DeNovix). 1 μg of RNA per sample was shipped to the Van Andel Institute Genomics core (333 Bostwick Ave. NE, Grand Rapids, MI 49,503) for RNA analysis.
RNAseq data was aligned using STAR (v 2.7.2b) and quantified using RSEM (v 1.3.3). Differential expression (DE) was performed using the diffExp function in the OCTAD package with edgeR as the method for DE. The cutoff for significance was chosen as absolute value of log2FoldChange > 1 and padj < 0.05.
Raw counts of individual samples were used to generate normalized log2 counts with edgeR (v 4.6.2). The expression values per gene were standardized using ggplot2 (v 3.5.2). Only genes found in either list of DE genes were used subsequently. Arrangement of samples (columns) was ordered after treatment groups and plotted using pheatmap (v 1.0.13). PCA analysis was also conducted using ggplot2 (v 3.5.2).
Analyses were performed in RStudio (v 2025.05.0-496) based on the tidyverse (v 2.0.0) package. The overview (DE genes) was created using EnhancedVolcano (v 1.26.0) in combination with dplyr (v 1.1.4). Gene information was obtained through org.Hs.eg.db (v 3.21.0), the enrichment was done using clusterProfiler (v 4.16.0), DOSE (v 4.2.0), and enrichplot (v 1.28.1). Visualization of highlighted pathways was done using ggrepel (v 0.9.6) and ggplot2 (v 3.5.2).
OVCAR3, OVCAR8, and PANC1 cells were plated in 24-well plates (Corning, Cat# 3526) with 1.5 × 10 cells per well and treated with a dose corresponding to 7 × IC, respectively. Durations of treatment were either 0 h, 4 h, or 24 h, and the DMSO concentration between samples was normalized. The RNA was isolated and purified using a RNeasy Mini Kit (Qiagen, Cat# 74,104). RNA amounts were quantified with a DS-11 Spectrophotometer (DeNovix). 1 μg of RNA per sample was converted into cDNA using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Cat# 4,374,966) in combination with a MiniAmp Plus Thermal Cycler (Applied Biosystems). Subsequently, qRT-PCR was performed with 10 ng of cDNA and 250 nM of primers per sample, using the Power SYBR Green PCR Master Mix (Applied Biosystems, Cat# 4,367,659). Runs were performed with a Bio-Rad C1000 Thermal Cycler equipped with a CFX96 Optical PCR Module (both Bio-Rad).
Volume of 10 μL, Lid: 105 °C; 50 °C 2:00 min > 95 °C, 10:00 min > 95 °C, 0.25 min > 60 °C, 1:00 min > Plate read > Go to 3, Repeat 39x.