p32-sphingosine-activated protein kinase (EC50 = 8 ± 3 μM)
p18-sphingosine-activated protein kinase (Activates at 20-100 μM) [1]
Protein kinase C (PKC) [2]
Protein phosphatase 2A (PP2A) [4]

The p32-sphingosine-activated protein responds to low doses of D-erythro-Sphingosine, with first activation observed at 2.5 μM and maximal activity observed at 10 -20 μM. The potential of sphingosine relative to other sphingosine stereomers, and the preference for sphingosine over hydroisosphingosine [1]. D-erythro-Sphingosine inhibits protein regulatory C in vitro[2]. D-erythro-Sphingosine has been demonstrated to inhibit linked polypeptide C that impacts cell regulation and numerous signal transductions, and demonstrates anti-tumor effect in a range of cells [3].

---

D-erythro-Sphingosine activated a p32-sphingosine-activated protein kinase in Jurkat T cell cytosol, causing an approximately 4-fold increase in phosphorylation of a p32 protein with initial activation observed at 2.5 μM and peak activity at 10-20 μM. [1]
It activated a p18-sphingosine-activated protein kinase, inducing phosphorylation of a p18 substrate at higher concentrations (20-100 μM), resulting in a 5-fold increase in phosphorylation at 100 μM. [1]
The activation of p32 kinase showed a modest specificity for D-erythro-sphingosine over its other stereoisomers (L-erythro, D-threo, L-threo). [1]
The activation of p18 kinase was specific to the erythro isomers of sphingosine; the threo isomers were largely inactive. [1]
D-erythro-Sphingosine showed a preference over its dihydro analog (dihydrosphingosine) in activating both p32 and p18 kinases. [1]
The sphingosine-induced phosphorylation of p32 and p18 was inhibited by oleic acid with an IC50 of approximately 20 μM. [1]
Both synthetic D-erythro-sphingosine and commercial sphingosine preparation potently inhibited purified human platelet protein kinase C (PKC) activity in a vesicle-based assay, with complete inhibition observed at 50 μM. [2]
In a mixed micellar assay using Triton X-100/phosphatidylserine/diacylglycerol, both synthetic and commercial D-erythro-sphingosine inhibited purified PKC with 50% inhibition occurring at a concentration of ~6 mol%. [2]
D-erythro-sphingosine inhibited γ-thrombin-induced phosphorylation of the 40 kDa protein (a PKC substrate) in intact human platelets. [2]
D-erythro-sphingosine inhibited human platelet aggregation induced by 8 nM γ-thrombin in a dose-dependent manner, with 50% inhibition occurring at approximately 10 μM. [2]
D-erythro-sphingosine inhibited ATP secretion from human platelets induced by 8 nM γ-thrombin. [2]
The inhibitory effects of synthetic and commercial D-erythro-sphingosine on PKC activity, platelet 40 kDa protein phosphorylation, aggregation, and secretion were equipotent. [2]
N,N-dimethylsphingosine (DMS) also inhibited purified PKC but was not found to be a contaminant in the commercial sphingosine preparation used. [2]
In cultured cerebellar granular neurons (CGNs), treatment with D-erythro-Sphingosine (DES, 10 nM) downregulated the level of tyrosine-307 phosphorylated PP2A (pY307) after 5-20 minutes of exposure. [4]
Treatment with DES (10 nM) also decreased the level of demethylated Leu-309 PP2A (DML309) after 20 minutes of application. [4]
PP2A activity assays demonstrated that DES application increased PP2A activity during a 5-20 minute exposure. [4]
DES treatment markedly reduced the phosphorylation level of collapsin response mediator protein-2 (pCRMP2) at 5-20 minutes. [4]
In neurite growth assays, pre-incubation of culture plates with inhibitory substrates (CSPGs or MAG) significantly decreased neurite outgrowth. Treatment with DES (10 nM) for 48 hours remarkably reversed this neurite outgrowth failure. [4]

In a rat model of spinal cord injury (SCI), chronic application of D-erythro-Sphingosine (DES, 1 μM solution) via a subcutaneous osmotic pump for 7 days enhanced PP2A activity around the lesion site. [4]
DES treatment significantly decreased the levels of pY307 and pCRMP2 in the injured spinal cord tissue compared to vehicle-treated rats. [4]
Immunofluorescence analysis of spinal cord white matter showed reduced pY307 and pCRMP2 signals after DES treatment. [4]
Anterograde tracing with biotinylated dextran amine (BDA) 32 days post-SCI revealed that DES treatment promoted axon sprouting, with thin sprouts extending into nearby gray matter and caudal to the lesion, unlike vehicle-treated controls. [4]
DES-treated rats showed significant improvement in locomotor function recovery from day 7 to day 28 after SCI, as evaluated by BBB (Basso, Beattie, Bresnahan) and CBS (Combined Behavioral Score) scales. [4]

A protein kinase assay was performed in a total volume of 50 μl containing 5 μl of a sphingosine suspension (or ethanol solution as control), 5 μl of other effectors, 20 μl of a Mg²⁺/ATP solution (containing 100 mM Tris/HCl pH 7.4, 25 mM MgCl₂, 62.5 μM ATP, and 62.5 μCi/ml [γ-³²P]ATP), and 20 μl of diluted Jurkat T cell cytosol (8–12 μg of protein per sample). [1]
The reaction mixture was incubated, and protein phosphorylation was assessed. [1]
Protein kinase C (PKC) activity was assayed using two methods. In the vesicle assay, the enzyme activity was measured in the presence of phosphatidylserine (PS) and dioleoylglycerol (DAG). [2]
In the mixed micellar assay, PKC activity was assayed using Triton X-100 mixed micelles containing PS and DAG. [2]
For inhibition studies, various concentrations of D-erythro-sphingosine, commercial sphingosine, or N,N-dimethylsphingosine were included in the reaction mixtures. Enzyme activity was expressed as a percentage of the activity measured without added inhibitor. [2]
PP2A activity was measured using a commercial protein phosphatase activity assay kit. Endogenous free phosphate was removed from cell supernatants or tissue extracts. [4]
Protein samples were incubated with a chemically synthesized phosphopeptide substrate optimal for PP2A, in a buffer optimized for PP2A activity while inhibiting cation-dependent phosphatases PP2B and PP2C. The reaction proceeded for 30 minutes at 33°C. [4]
The amount of phosphate released from the substrate was determined by measuring the absorbance of a molybdate-malachite green-phosphate complex at 630 nm. [4]

Jurkat T cells were grown in RPMI-1640 medium supplemented with fetal bovine serum and maintained at a density of 2×10⁵ to 1.2×10⁶ cells/mL. [1]
Cytosolic fractions were prepared from these cells. [1]
To assess the effects of lipids on protein phosphorylation, cytosolic extracts were incubated with various concentrations of D-erythro-sphingosine, its stereoisomers, dihydrosphingosine isomers, or fatty acids like oleic acid in the kinase assay system described above. [1]
Changes in the phosphorylation levels of specific substrate proteins (p32 and p18) were quantified. [1]
Washed human platelets were prepared from the blood of healthy volunteers. [2]
For aggregation and secretion assays, platelets were pre-incubated with indicated concentrations of D-erythro-sphingosine (delivered in ethanol) and then stimulated with 8 nM γ-thrombin. Aggregation and ATP secretion were monitored. [2]
For the 40 kDa substrate phosphorylation assay, platelet pellets were stimulated with 8 nM γ-thrombin in the presence or absence of different preparations of sphingosine. Phosphorylated proteins were separated by SDS-PAGE, the 40 kDa band was excised, and incorporated ³²P was counted. [2]
Primary cerebellar granular neurons (CGNs) were cultured from 1- to 3-day-old rat pups. Dissociated tissues were digested and seeded on poly-D-lysine pre-coated culture plates. [4]
In some experiments, plates were pre-coated with inhibitory substrates (CSPGs or MAG) for 4 hours at 37°C. [4]
Neurons were treated with D-erythro-Sphingosine (DES, 10 nM) or other agents 2-4 hours after plating. [4]
For protein analysis, neurons were lysed after 24 hours of culture or at specified time points after treatment for Western blotting. [4]
For neurite outgrowth analysis, neurons were treated for 48 hours, then fixed and stained with βIII-tubulin antibody. Neurite length was measured using image analysis software. [4]
For PP2A activity measurement, cell supernatants were collected at specified time points after treatment. [4]

A moderate spinal cord injury (SCI) was induced in adult Sprague-Dawley rats at the T11 level using a weight-drop impactor. [4]
Immediately after SCI, a subcutaneous osmotic pump was implanted close to the injury site. [4]
The pump was filled with either saline (vehicle) or a solution of D-erythro-Sphingosine (DES, 1 μM). [4]
A catheter connected the pump to the subdural space on the dorsal aspect of the spinal cord at the injury center. The pump delivered the solution continuously. [4]
For chronic treatment assessing functional recovery, the pump was removed 7 days after surgery. Behavioral tests (BBB, CBS) were performed periodically up to 28 days post-injury. [4]
For tissue analysis, rats were sacrificed at specified time points (e.g., 7 days post-injury for Western blot and immunofluorescence). [4]

[1]. Regulation of sphingosine-activated protein kinases: selectivity of activation by sphingoid basesand inhibition by non-esterified fatty acids. Biochem J. 1993 Sep 15;294 ( Pt 3):699-703.

[2]. Protein kinase C and platelet inhibition by D-erythro-Sphingosine: comparison with N,N-dimethylsphingosine and commercial preparation. Biochem Biophys Res Commun. 1990 Oct 30;172(2):683-91.

[3]. A concise synthesis of a promising protein kinase C inhibitor: D-erythro-Sphingosine. Arch Pharm Res. 2007 Jan;30(1):22-7.

[4]. Protein phosphatase 2A (PP2A) activation promotes axonal growth and recovery in the CNS. J Neurol Sci. 2015 Dec 15;359(1-2):48-56.

Sphingosine is a sphingo-4-ene with a trans configuration. It is a metabolite in both humans and mice. It is the conjugate base of sphingosine (1+) and the enantiomer of L-erythrosphingosine. Sphingosine is an amino alcohol with a long unsaturated hydrocarbon chain. Sphingosine and its derivatives are the major bases of mammalian sphingolipids. (Dorland, 28th edition) Sphingosine has been reported in Rehmannia glutinosa, soybean, and several other organisms with relevant data. Sphingosine is an amino alcohol with a long unsaturated hydrocarbon chain. Sphingosine and its derivatives are the major bases of mammalian sphingolipids. (Dorland, 28th edition) See also: L-erythrosphingosine (note moved to). D-erythrosphingosine is a bioactive sphingolipid that can activate specific protein kinases in cell-free systems. [1]
Its action is stereospecific, and the erythroform is crucial for activity, especially for p18 kinase. [1]
This study distinguished two different sphingosine-activated protein kinases (p32-SAPK and p18-SAPK) based on substrate specificity, activation potency, and stereochemical preference. [1]
Fatty acids (such as oleic acid) can inhibit sphingosine-induced phosphorylation, revealing the possible antagonistic effects of different lipid messengers in regulating protein kinase activity. [1] D-erythrosine is a potent and specific inhibitor of protein kinase C. [2] The commercially available sphingosine formulations used in this study mainly contain the D-erythroform stereoisomer, and no N,N-dimethylsphingosine contamination was detected. [2] This study validated the practicality of well-defined commercially available sphingosine as a pharmacological inhibitor of PKC in in vitro and cell studies. [2] In this study, D-erythrosine (DES) was used as a classic agonist of protein phosphatase 2A (PP2A). [4] This study identified a signaling pathway in which inhibitory substrates (CSPG, MAG) activate EGFR, which then phosphorylates and inactivates the Y307 site of PP2A. Inactivated PP2A cannot dephosphorylate CRMP2, leading to impaired axonal growth. [4]
DES works by activating PP2A, promoting CRMP2 dephosphorylation, thereby promoting axonal regeneration and functional recovery after spinal cord injury. [4]
This suggests that pharmacological activation of PP2A, such as with D-erythrosphospirin, may be a potential strategy for treating axonal injury of the central nervous system. [4]

C₁₈H₃₇NO₂

299.49

299.282

C, 72.19; H, 12.45; N, 4.68; O, 10.68

123-78-4

D-erythro-Sphingosine hydrochloride;2673-72-5;D-erythro-Sphingosine-d7;1246304-34-6

5280335

White to off-white solid powder

0.9±0.1 g/cm3

445.9±45.0 °C at 760 mmHg

81-82ºC

223.5±28.7 °C

0.0±2.4 mmHg at 25°C

1.489

6.4

3

3

15

21

231

2

CCCCCCCCCCCCC/C=C/[C@@H](O)[C@@H](N)CO

WWUZIQQURGPMPG-KRWOKUGFSA-N

InChI=1S/C18H37NO2/c1-2-3-4-5-6-7-8-9-10-11-12-13-14-15-18(21)17(19)16-20/h14-15,17-18,20-21H,2-13,16,19H2,1H3/b15-14+/t17-,18+/m0/s1

2S-amino-4E-octadecene-1,3R-diol

Erythrosphingosine erythro-C18-Sphingosine trans-4-Sphingenine(−)-Sphingosine D-erythro-Sphingosine C-18 Sphingosine (d18

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~50 mg/mL (~166.95 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (8.35 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

---

Solubility in Formulation 2: ≥ 2.5 mg/mL (8.35 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

---

 (Please use freshly prepared in vivo formulations for optimal results.)