RBM1-151 is a 1-deoxy derivative and vinyl analog of RBM14-C12, serving as a fluorescent substrate for amidases (Ex/Em = 355/460 nm). It is hydrolyzed by acidic ceramidase (AC) (appKm = 7.0 μM; appVmax = 99.3 nM/min), N-acylethanolamine hydrolyzing acid amidase (appKm = 0.73 μM; appVmax = 0.24 nM/min), and fatty acid amide hydrolase (FAAH) (appKm = 3.6 μM; appVmax = 7.6 nM/min), but is not hydrolyzed by other ceramidases. RBM1-151 is useful for studying the basic biology of lipid amidase functions and for potential diagnostic/prognostic assessments in disorders involving AC, NAAA, or FAAH dysregulation, such as Farber disease and cancer.

RBM1-151 (20 μM, 1 hour) is hydrolyzed to form RBM1-151-NH2 solely by acidic ceramidase in a cell-free system (using A375/AC cell lysate). In acidic buffer, RBM1-151 (0-20 μM, 30 minutes) is hydrolyzed by A375/AC cell lysate (20 μg), with kinetic parameters calculated via Michaelis-Menten analysis showing an app Km of 7.0 μM and an app Vmax of 99.3 nM/min. In HEK293/NAAA cell lysate (10 μg, overexpressing NAAA), RBM1-151 (5 μM, 3 hours) exhibits higher hydrolysis compared to HEK293/mock lysate, as indicated by fluorescence product. In Farber disease (FD) cells (lacking AC), RBM1-151 (20 μM, 3 hours) yields minimal fluorescence, whereas fluorescence is significantly higher in FD/AC cells (overexpressing AC) in whole cells. In A375/AC cells (overexpressing AC), RBM1-151 (20 μM, 3 hours) shows greater hydrolysis than in A375/WT cells. Guidelines: 1. Cell Preparation: 1.1 For suspension cells (e.g., AML cell lines: MM-6, HL-60), culture cells in RPMI-1640 medium with 20% FBS; adjust density to 2 × 10^4 cells per well before plating. 1.2 For adherent cells (e.g., melanoma cell lines: A375/AC, C8161; HEK293), use Dulbecco modified Eagle medium (high glucose) with 10% FBS and 1% penicillin/streptomycin; seed 2 × 10^4 cells per well in a 96-well plate and incubate overnight for adherence. Note: For antibiotic-selective cell lines (e.g., A375/AC using blasticidin and hygromycin), remove antibiotics before experimentation to prevent interference. 2. RBM1-151 Incubation and Signal Detection: 2.1 Add 50 μL of 20 μM RBM1-151 working solution (prepared in medium with 20% FBS, final concentration 20 μM) to each well. 2.2 Incubate at 37°C with 5% CO2 for 1 hour. 2.3 Add 25 μL of 100% methanol to each well to stop the reaction. 2.4 Immediately add 100 μL of NaIO4 solution (2.5 mg/mL, prepared in 100 mM glycine-NaOH buffer, pH 10.6). 2.5 Incubate in the dark at 37°C with 5% CO2 for 30 minutes. 2.6 Measure fluorescence signals using a microplate reader, subtracting background signals from wells containing only medium and RBM1-151 without cells.