C16-Ceramide is a natural small molecule that activates/agonizes p53 through direct selective binding.

Physicochemical Properties

Molecular Formula	C34H67NO3
Molecular Weight	537.90068
Exact Mass	537.512
CAS #	4201-58-5
Related CAS #	C16-Ceramide-d31;852043-41-5;C16-Ceramide-13C16;C16-Ceramide-d9;2260669-51-8
PubChem CID	5283564
Appearance	White to off-white solid powder
LogP	10.403
Hydrogen Bond Donor Count	3
Hydrogen Bond Acceptor Count	3
Rotatable Bond Count	30
Heavy Atom Count	38
Complexity	508
Defined Atom Stereocenter Count	2
SMILES	CCCCCCCCCCCCCCCC(=O)N[C@@H](CO)[C@@H](/C=C/CCCCCCCCCCCCC)O
InChi Key	YDNKGFDKKRUKPY-TURZORIXSA-N
InChi Code	InChI=1S/C34H67NO3/c1-3-5-7-9-11-13-15-17-19-21-23-25-27-29-33(37)32(31-36)35-34(38)30-28-26-24-22-20-18-16-14-12-10-8-6-4-2/h27,29,32-33,36-37H,3-26,28,30-31H2,1-2H3,(H,35,38)/b29-27+/t32-,33+/m0/s1
Chemical Name	N-[(E,2S,3R)-1,3-dihydroxyoctadec-4-en-2-yl]hexadecanamide
HS Tariff Code	2934.99.9001
Storage	Powder-20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month Note: This product requires protection from light (avoid light exposure) during transportation and storage.
Shipping Condition	Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Biological Activity

ln Vitro	C16-ceramide interacts with p53 within its core domain. p53 complexes with natural C16-Ceramide in the cell [1]. C16-Ceramide (2.5-50 μM; 0-48 h) significantly reduced HCT116 cell viability in a time and concentration-dependent manner [2]. C16-Ceramide (12 μM; 48 h) causes apoptosis in HCT116 cells via Btf (Bcl-2-associated transcription factor) [2]. C16-ceramide (12 μM; 0-6 h) and Btf expression enhance p53 and BAX expression. C16-ceramide suppresses Mdm2 expression via Btf[2].
Cell Assay	Cell Viability Assay[2] Cell Types: HCT116 cells Tested Concentrations: 2.5, 5, 10, 12, 20, 50 µM Incubation Duration: 0-48 h Experimental Results: Strongly diminished cell viability in a time- and concentration-dependent manner. Western Blot Analysis[2] Cell Types: HCT116 cells Tested Concentrations: 12 μM Incubation Duration: 1, 3 and 6 h Experimental Results: Increased PARP cleavage, diminished pro-caspase 3. diminished the levels of stratifin and stathmin, increased the expression of prohibitin and Btf. RNAi-mediated Btf depletion also partially inhibited BAX expression after the treatment. Dramatically diminished luciferase activity and Mdm2 protein expression levels.
References	[1]. C16-ceramide is a natural regulatory ligand of p53 in cellular stress response. Nat Commun. 2018 Oct 8;9(1):4149. [2]. The proapoptotic C16-ceramide-dependent pathway requires the death-promoting factor Btf in colon adenocarcinoma cells. J Proteome Res. 2009 Oct;8(10):4810-22.
Additional Infomation	N-hexadecanoylsphingosine is a N-acylsphingosine in which the ceramide N-acyl group is specified as hexadecanoyl (palmitoyl). It has a role as a Mycoplasma genitalium metabolite and a human blood serum metabolite. It is a N-acylsphingosine, a Cer(d34:1) and a N-palmitoyl-sphingoid base. It is functionally related to a hexadecanoic acid. N-Palmitoylsphingosine has been reported in Junceella juncea and Trypanosoma brucei with data available.

Solubility Data

Solubility (In Vitro)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Solubility (In Vivo)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples. Injection Formulations (e.g. IP/IV/IM/SC) Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline) *Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution. Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil) Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals). Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)] *Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution. Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline) Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline) Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline) Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil) Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Oral Formulations Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium) Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals). Oral Formulation 3: Dissolved in PEG400 Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose Oral Formulation 6: Mixing with food powders Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	1.8591 mL	9.2954 mL	18.5908 mL
	5 mM	0.3718 mL	1.8591 mL	3.7182 mL
	10 mM	0.1859 mL	0.9295 mL	1.8591 mL

*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.