Thiamet G is a novel potent, and selective O-GlcNAcase (OGA) inhibitor with Ki of 21 nM, it displayed 37,000-fold selectivity over human lysosomal–hexosaminidase. Oligomerization of tau is a key process contributing to the progressive death of neurons in Alzheimer's disease. Tau is modified by O-linked N-acetylglucosamine (O-GlcNAc), and O-GlcNAc can influence tau phosphorylation in certain cases. O-GlcNAc also inhibits thermally induced aggregation of an unrelated protein, TAK-1 binding protein, suggesting that a basic biochemical function of O-GlcNAc may be to prevent protein aggregation. These results also suggest O-GlcNAcase as a potential therapeutic target that could hinder progression of Alzheimer's disease. Thiamet G was extremely stable in aqueous solution. In nerve growth factor (NGF)-differentiated PC-12 cells, thiamet G significantly increased cellular O-GlcNAc levels with EC50 value of 30 nM in a dose dependent way.

Physicochemical Properties

Molecular Formula	C9H16N2O4S
Molecular Weight	248.3
Exact Mass	248.083
Elemental Analysis	C, 43.54; H, 6.50; N, 11.28; O, 25.77; S, 12.91
CAS #	1009816-48-1
Related CAS #	1009816-48-1
Appearance	White to light yellow solid
Density	1.8±0.1 g/cm3
Boiling Point	483.2±55.0 °C at 760 mmHg
Flash Point	246.0±31.5 °C
Vapour Pressure	0.0±2.8 mmHg at 25°C
Index of Refraction	1.729
LogP	-0.09
SMILES	S1/C(=N\C([H])([H])C([H])([H])[H])/N([H])C2([H])[C@]1([H])OC([H])(C([H])([H])O[H])[C@]([H])(C2([H])O[H])O[H]
InChi Key	PPAIMZHKIXDJRN-FMDGEEDCSA-N
InChi Code	InChI=1S/C9H16N2O4S/c1-2-10-9-11-5-7(14)6(13)4(3-12)15-8(5)16-9/h4-8,12-14H,2-3H2,1H3,(H,10,11)/t4-,5-,6-,7-,8-/m1/s1
Chemical Name	(3aR,5R,6S,7R,7aR)-2-(Ethylamino)-3a,6,7,7a-tetrahydro-5-(hydroxymethyl)-5H-pyrano[3,2-d]thiazole-6,7-diol
Synonyms	Thiamet-G;Thiamet G
HS Tariff Code	2934.99.03.00
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

Targets	Human OGA (Ki = 20 nM)
ln Vitro	In ATDC5 cells, thiamet G (1 μM) dramatically enhances the accumulation of O-GlcNAcylated protein. The buildup of O-GlcNAc brought on by thiamet G also significantly increased these MMPs' activity. JNK, ERK, and p38 are all phosphorylated when exposed to 1 μM of thiamet G, but not Akt[2]. Cell viability is not greatly affected by thiamet G (0.1–10 μM). Microtubule dynamics are changed and tau phosphorylation is decreased by thiamet G [3].
ln Vivo	At 500 mg/kg/d, thiamet G decreases neurodegeneration and raises tau and global O-GlcNAc. In this transgenic model, thiamet G treatment prevented tau-driven neurodegeneration and increased motor neurons by 1.4 times. Therefore, Thiamet G treatment was ineffective in mice devoid of the P301L transgene, suggesting that Thiamet G treatment is only effective in preventing neurodegeneration and weight loss when the P301L transgene is present. O-GlcNAc is elevated in the brain and spinal cord tissue of Thiamet G-treated mice [1]. O-GlcNAc levels in the brain, liver, and knee joints of C57BL/6 mice are dose-dependently increased by thiamet G (20 mg/kg, i.p.) [2].
Enzyme Assay	All enzymatic assays are performed in triplicate at 37°C using 4-methylumbelliferyl N-acetyl-β-d-glucosaminide dehydrate as substrate. 1 nM of purified OGA is incubated with the compounds for 5 min, and then 0.2 mM of the substrate is added. The liberation of 4-methylumbellifery is monitored by kinetic reading at excitation/emission 355/460 nm using a Tecan M200 plate in a mode of 60 s/cycle and 15 cycles in total.[3]
Cell Assay	Jurkat cells are seeded at 6000 cells/well in a 96-well plate, and 12 h later, cells are treated with compounds for the indicated time. Cell viability is determined by XTT assay [3].
Animal Protocol	For the Thiamet G dose dependence study, six 23-day-old male C57BL/6 mice receive single intraperitoneal injections of either 0, 10, 20, 100, 200, or 500 mg/kg of Thiamet G dissolved in PBS and then are euthanized 8 h later to evaluate the O-GlcNAc levels in different tissues (brain, liver, muscle, and knee). The time of sacrifice is chosen on the basis of previously published data on Thiamet G in rodents, which demonstrates that the peak level of O-GlcNAc proteins following administration of the drug is achieved after 8-10 h. Tissues are collected immediately after sacrifice, flash-frozen in liquid nitrogen, and stored at −80°C until required for use [2]. Dissolved in water; Healthy Sprague-Dawley rats.; p.o. or i.v.
References	[1]. Increasing O-GlcNAc slows neurodegeneration and stabilizes tau against aggregation. Nat Chem Biol. 2012 Feb 26;8(4):393-9. [2]. The increase in O-linked N-acetylglucosamine protein modification stimulates chondrogenic differentiation both in vitro and in vivo. J Biol Chem. 2012 Sep 28;287(40):33615-28. [3]. Thiamet-G-mediated inhibition of O-GlcNAcase sensitizes human leukemia cells to microtubule-stabilizing agent NSC 125973. Biochem Biophys Res Commun. 2014 Oct 24;453(3):392-7.

Solubility Data

Solubility (In Vitro)

DMSO:50 mg/mL (201.4 mM) Water:50 mg/mL (201.4 mM) Ethanol:12 mg/mL (48.3 mM)

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 2.08 mg/mL (8.38 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 20.8 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: ≥ 2.08 mg/mL (8.38 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 20.8 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: ≥ 2.08 mg/mL (8.38 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 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly. Solubility in Formulation 4: Saline: 30 mg/mL Solubility in Formulation 5: 50 mg/mL (201.37 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	4.0274 mL	20.1369 mL	40.2739 mL
	5 mM	0.8055 mL	4.0274 mL	8.0548 mL
	10 mM	0.4027 mL	2.0137 mL	4.0274 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.