Neoseptin-3 is a novel and potent agonist of mouse TLR4 (mTLR4) Toll-like receptor 4/myeloid differentiation factor 2 (mTLR4/MD-2) with an EC50 of 18.5 μM.

Physicochemical Properties

Molecular Formula	C29H34N2O4
Molecular Weight	474.591268062592
Exact Mass	474.251
CAS #	1622863-21-1
PubChem CID	77461013
Appearance	White to off-white solid powder
LogP	5.6
Hydrogen Bond Donor Count	3
Hydrogen Bond Acceptor Count	5
Rotatable Bond Count	11
Heavy Atom Count	35
Complexity	662
Defined Atom Stereocenter Count	1
SMILES	C(C1C=CC(N)=C(CCC2C=CC(O)=CC=2)C=1)(=O)N[C@H](C(=O)OC(C)(C)C)CCC1C=CC=CC=1
InChi Key	OACODUCFPHHCIH-SANMLTNESA-N
InChi Code	InChI=1S/C29H34N2O4/c1-29(2,3)35-28(34)26(18-12-20-7-5-4-6-8-20)31-27(33)23-14-17-25(30)22(19-23)13-9-21-10-15-24(32)16-11-21/h4-8,10-11,14-17,19,26,32H,9,12-13,18,30H2,1-3H3,(H,31,33)/t26-/m0/s1
Chemical Name	tert-butyl (2S)-2-[[4-amino-3-[2-(4-hydroxyphenyl)ethyl]benzoyl]amino]-4-phenylbutanoate
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
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	Neoseptin 3 possesses an EC50 of 18.5 μM, making it an agonist of Toll-like receptor 4/myeloid differentiation factor 2 (mTLR4/MD-2). Macrophages produce TNFα in response to neoseptin-3, and this response is concentration-dependent. In accordance with the activation of MAPK and classical NF-κB signaling, neoseptin-3 causes the phosphorylation of IκB kinases α (IKKα), IKKβ, p38, c-Jun N-terminal kinase (JNK), and ERK, as well as the degradation of IκBα. Neoseptin-3 also causes an increase in the phosphorylation of IRF3 and TANK-binding kinase 1 (TBK1)[1].
Enzyme Assay	HEK293T cells are transfected with an NF-κB-dependent luciferase reporter plasmid and clones with stable expression are selected by culture in DMEM containing puromycin. Cells are cotransfected with constructs for mouse or human TLR4 plus mouse or human MD-2, and 2 d later are stimulated with 50 μM Neoseptin-3 or 1 μg/mL LPS for 6 h. Cells are lysed, and luciferase activity is measured[1].
Cell Assay	Cells are seeded onto 96-well plates at 1×105 cells per well and stimulated with Neoseptin-3 [dissolved in DMSO; final DMSO concentrations (≤0.2%) are kept constant in all experiments] for 4 h. Mouse TNFα, IL-6, or IFN-β, or human TNFα in the supernatants are measured by ELISA kits according to the manufacturer’s instructions. Unless otherwise indicated, mouse cells are from wild-type C57BL/6J mice[1].
References	[1]. TLR4/MD-2 activation by a synthetic agonist with no similarity to LPS. Proc Natl Acad Sci U S A. 2016 Feb 16;113(7):E884-93.

Solubility Data

Solubility (In Vitro)

DMSO : ≥ 100 mg/mL (~210.71 mM)

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.27 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 (5.27 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in 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 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly. 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. Solubility in Formulation 3: ≥ 2.5 mg/mL (5.27 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.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	2.1071 mL	10.5354 mL	21.0708 mL
	5 mM	0.4214 mL	2.1071 mL	4.2142 mL
	10 mM	0.2107 mL	1.0535 mL	2.1071 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.