TFMB-(S)-2-HG is a potent inhibitor of TET2 (ten-eleven translocation 2), a 5'-methylcytosine hydroxylase that catalyzes the oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), playing a critical role in DNA demethylation and epigenetic regulation. By inhibiting TET2, (S)-2-HG contributes to the hypermethylation phenotype observed in IDH-mutant cancers, blocking the normal DNA demethylation process and altering gene expression patterns that promote tumorigenesis. Additionally, TFMB-(S)-2-HG significantly inhibits the EglN prolyl hydroxylases (PHDs), which are key regulators of hypoxia-inducible factor (HIF) stability. Under normoxic conditions, PHDs hydroxylate HIF-α subunits, marking them for degradation; inhibition of PHDs by (S)-2-HG leads to HIF-α stabilization and activation of hypoxia-responsive genes, which can contribute to tumor progression and metabolic reprogramming. TFMB-(S)-2-HG also downregulates Wnt3a and β-catenin (intranuclear) protein expression. The Wnt/β-catenin signaling pathway is a critical regulator of cell proliferation, differentiation, and stem cell maintenance, and its dysregulation is implicated in various cancers. By downregulating this pathway, (S)-2-HG may exert additional effects on cellular growth and differentiation. Furthermore, TFMB-(S)-2-HG inhibits osteogenic differentiation of cells, an effect likely mediated through its inhibition of TET2 and modulation of epigenetic and signaling pathways. The compound's multi-targeted activity—spanning epigenetic enzymes (TET2), oxygen-sensing pathways (PHDs), and developmental signaling cascades (Wnt/β-catenin)—makes it a powerful tool for dissecting the complex, pleiotropic effects of the oncometabolite (S)-2-HG in various cellular contexts.

In cell-free enzymatic assays, TFMB-(S)-2-HG serves as a potent inhibitor of TET2 and EglN prolyl hydroxylases. The compound demonstrates significant inhibitory activity against TET2, the 5'-methylcytosine hydroxylase, thereby blocking the conversion of 5-methylcytosine to 5-hydroxymethylcytosine. It also exhibits potent inhibition of EglN prolyl hydroxylases, enzymes that regulate HIF-α stability. In addition to its enzyme inhibitory activities, TFMB-(S)-2-HG downregulates Wnt3a and β-catenin (intranuclear) protein expression. This downregulation of Wnt/β-catenin signaling may contribute to the compound's effects on cell differentiation and proliferation. The compound also inhibits osteogenic differentiation of cells, further demonstrating its impact on cellular differentiation programs. TFMB-(S)-2-HG is widely employed in cancer biology and epigenetics research to model IDH mutation-driven metabolic and transcriptional changes. Its ability to recapitulate the effects of (S)-2-HG accumulation in a controlled manner allows researchers to study the downstream consequences of oncometabolite activity, including DNA methylation changes, altered gene expression, and metabolic reprogramming. The compound is also used to investigate the role of (S)-2-HG in the regulation of HIF signaling and the Wnt/β-catenin pathway, providing insights into the mechanisms by which IDH mutations drive tumorigenesis. The cell-permeable nature of TFMB-(S)-2-HG enables its use in a wide range of in vitro experimental systems, from purified enzyme assays to complex cell culture models. Typical concentrations used in culture range from 0.5 to 20 mM. The compound has potential for research into acute myeloid leukemia (AML) and other cancers where IDH mutations and 2-HG accumulation play a pathogenic role. In cellular assays, TFMB-(S)-2-HG is used to investigate the effects of (S)-2-HG on various cell types, including cancer cells, stem cells, and differentiated cells. The compound is cell-permeable, allowing it to be taken up by cells and converted intracellularly to the active (S)-2-hydroxyglutarate metabolite. Typical concentrations used in culture range from 0.5 to 20 mM. In AML research, TFMB-(S)-2-HG is used to model the effects of IDH mutations on cellular differentiation, proliferation, and survival. Treatment of leukemic cells with TFMB-(S)-2-HG leads to inhibition of TET2 activity, resulting in DNA hypermethylation and altered gene expression patterns that recapitulate the epigenetic changes seen in IDH-mutant AML. The compound also inhibits EglN prolyl hydroxylases, leading to HIF-α stabilization and activation of hypoxia-responsive genes. Furthermore, TFMB-(S)-2-HG downregulates Wnt3a and β-catenin protein expression, affecting Wnt/β-catenin signaling and potentially influencing cell differentiation and stem cell maintenance. In studies of osteogenic differentiation, TFMB-(S)-2-HG has been shown to inhibit the differentiation of cells into osteoblasts, an effect that may be mediated through its inhibition of TET2 and modulation of epigenetic and signaling pathways. The compound is also used to study the role of (S)-2-HG in metabolic reprogramming, including alterations in cellular metabolism and energy production. By providing a controlled source of (S)-2-HG, TFMB-(S)-2-HG allows researchers to dissect the specific contributions of this oncometabolite to the complex phenotype of IDH-mutant cancers, including changes in DNA methylation, gene expression, cellular metabolism, and differentiation.

TFMB-(S)-2-HG is a cell-permeable ester prodrug designed to deliver (S)-2-hydroxyglutarate [(S)-2-HG] into cells and tissues for in vivo studies. In animal models, the compound is used to investigate the role of (S)-2-HG in tumorigenesis, epigenetic regulation, and metabolic reprogramming. The prodrug is administered to animals, where it is taken up by cells and converted intracellularly to the active (S)-2-HG metabolite. This allows researchers to study the effects of (S)-2-HG accumulation in vivo, mimicking the conditions seen in IDH-mutant cancers. TFMB-(S)-2-HG has been used in mouse models of AML to study the effects of (S)-2-HG on leukemogenesis, including its impact on hematopoietic stem cell differentiation, proliferation, and survival. The compound has also been used in models of other cancers where IDH mutations and 2-HG accumulation play a role. In vivo studies with TFMB-(S)-2-HG typically involve administration via intraperitoneal injection or oral gavage, depending on the experimental design and the desired route of administration. Following administration, the prodrug is rapidly taken up by tissues and converted to (S)-2-HG, leading to elevated levels of the oncometabolite in target organs. Researchers then assess the effects of (S)-2-HG on various endpoints, including tumor growth, epigenetic modifications, gene expression, and metabolic changes. The compound has been used to study the role of (S)-2-HG in the regulation of TET2 activity, HIF signaling, and the Wnt/β-catenin pathway in vivo. TFMB-(S)-2-HG is also used to investigate the effects of (S)-2-HG on normal tissue homeostasis and differentiation, such as its inhibition of osteogenic differentiation. By enabling the controlled elevation of (S)-2-HG in vivo, TFMB-(S)-2-HG provides a valuable tool for understanding the pathophysiological role of this oncometabolite and for evaluating potential therapeutic strategies targeting IDH-mutant cancers.

Cell-free assays with TFMB-(S)-2-HG typically involve the use of purified enzymes or cell lysates to assess its inhibitory activity against specific targets. For TET2 inhibition, the enzyme is incubated with a DNA substrate containing 5-methylcytosine and varying concentrations of TFMB-(S)-2-HG. TET2 activity is then measured by quantifying the conversion of 5-methylcytosine to 5-hydroxymethylcytosine using techniques such as mass spectrometry, ELISA, or dot blot analysis. The IC50 value for TET2 inhibition can be determined from these assays. For EglN prolyl hydroxylase inhibition, the enzyme is incubated with a HIF-α peptide substrate and varying concentrations of TFMB-(S)-2-HG. Hydroxylation of the peptide is measured using mass spectrometry or antibody-based detection methods, and the IC50 for inhibition is calculated. The compound's ability to downregulate Wnt3a and β-catenin protein expression can be assessed in cell-free systems using purified proteins or cell lysates, followed by Western blot analysis or ELISA. These cell-free assays are essential for characterizing the direct inhibitory effects of TFMB-(S)-2-HG on its molecular targets, independent of cellular uptake and metabolism. The compound is also used in cell-free systems to study its effects on enzyme kinetics and to compare its inhibitory potency against other 2-HG analogs or inhibitors. Typical concentrations used in these assays range from 0.5 to 20 mM, depending on the target and the experimental conditions. The results from these cell-free studies provide mechanistic insights into the oncometabolite activity of (S)-2-HG and help validate its role in the epigenetic and metabolic dysregulation observed in IDH-mutant cancers.

In cellular assays, TFMB-(S)-2-HG is typically added to cell culture media at concentrations ranging from 0.5 to 20 mM. The compound is cell-permeable and is taken up by cells, where it is converted intracellularly to the active (S)-2-hydroxyglutarate metabolite. Cells are treated with the compound for varying durations, typically 24 to 72 hours, depending on the experimental endpoint. Following treatment, cells are harvested and analyzed for various outcomes. TET2 activity is assessed by measuring the levels of 5-hydroxymethylcytosine (5hmC) in genomic DNA using techniques such as dot blot, ELISA, or mass spectrometry. Inhibition of TET2 by TFMB-(S)-2-HG results in decreased 5hmC levels and increased DNA methylation. EglN prolyl hydroxylase inhibition is assessed by measuring HIF-α protein levels via Western blot, as inhibition of PHDs leads to HIF-α stabilization. Wnt/β-catenin signaling is evaluated by measuring β-catenin protein levels in the nucleus and cytoplasm, as well as the expression of Wnt target genes using qPCR. Cell proliferation and viability are assessed using MTT, CellTiter-Glo, or trypan blue exclusion assays. Apoptosis is evaluated by flow cytometry using Annexin V/PI staining or by measuring caspase activity. Cell differentiation, such as osteogenic differentiation, is assessed using specific staining methods (e.g., Alizarin Red S) and by measuring the expression of differentiation markers via qPCR or Western blot. TFMB-(S)-2-HG is also used in studies of cellular metabolism, where changes in metabolite levels, oxygen consumption, and extracellular acidification are measured. These cellular assays are crucial for understanding the functional consequences of (S)-2-HG accumulation in various cell types and for modeling the effects of IDH mutations on cellular processes.

In vivo studies with TFMB-(S)-2-HG typically use mouse models, including xenograft or syngeneic tumor models, as well as genetically engineered mouse models of cancer. The compound is administered to animals via intraperitoneal (i.p.) injection or oral gavage, at doses that are determined based on preliminary pharmacokinetic and toxicity studies. The prodrug is designed for efficient cellular uptake and conversion to the active (S)-2-hydroxyglutarate metabolite. Following administration, blood and tissue samples are collected at various time points to measure the levels of TFMB-(S)-2-HG and (S)-2-HG using LC-MS/MS. Tumor growth is monitored by caliper measurements or bioluminescent imaging. At the end of the study, tumors and other organs are harvested for histological analysis, immunohistochemistry, and molecular analysis. TET2 activity in tumor tissues is assessed by measuring 5hmC levels in genomic DNA. HIF-α levels and Wnt/β-catenin signaling are evaluated by Western blot and immunohistochemistry. Gene expression changes are analyzed by RNA-seq or qPCR. The compound's effects on osteogenic differentiation can be assessed in vivo using bone formation assays or by measuring bone density and histomorphometry. These in vivo studies are essential for understanding the role of (S)-2-HG in tumorigenesis and for evaluating the potential of targeting 2-HG metabolism as a therapeutic strategy. The compound is also used in studies of AML, where it is administered to mouse models of the disease to assess its effects on leukemogenesis and disease progression. By providing a controlled source of (S)-2-HG in vivo, TFMB-(S)-2-HG enables researchers to dissect the complex, multi-faceted effects of this oncometabolite in a physiologically relevant context.

TFMB-(S)-2-HG is a prodrug of (S)-2-hydroxyglutarate [(S)-2-HG], designed to be cell-permeable and to enable efficient delivery of the oncometabolite into cells and tissues. The pharmacokinetic properties of the compound are determined by its prodrug nature and the subsequent metabolism to (S)-2-HG. Following administration, TFMB-(S)-2-HG is taken up by cells, where it is converted intracellularly to the active (S)-2-HG metabolite. The ester prodrug moiety is cleaved by intracellular esterases, releasing (S)-2-HG. This conversion is efficient, allowing for rapid elevation of (S)-2-HG levels in target tissues. The pharmacokinetic profile of TFMB-(S)-2-HG includes parameters such as bioavailability, half-life, and tissue distribution, which are evaluated in preclinical studies using LC-MS/MS analysis of the prodrug and its active metabolite. The compound's lipophilicity, conferred by the TFMB ester moiety, enhances its membrane permeability and cellular uptake compared to the free acid form of (S)-2-HG. This property is critical for its effectiveness as a cell-permeable precursor. The pharmacokinetics of TFMB-(S)-2-HG may vary depending on the route of administration (e.g., intraperitoneal injection vs. oral gavage) and the formulation used. Studies typically measure the concentration of TFMB-(S)-2-HG and (S)-2-HG in plasma and tissues over time to determine the Cmax, Tmax, half-life, and area under the curve (AUC). The compound's ability to deliver (S)-2-HG to target tissues, including tumors and bone marrow, is a key consideration in its use for in vivo studies. The pharmacokinetic data obtained from these studies are essential for designing appropriate dosing regimens and for interpreting the results of efficacy and toxicity studies. The compound's metabolism and clearance pathways are also characterized to understand its elimination from the body. Overall, the pharmacokinetic properties of TFMB-(S)-2-HG are optimized for its use as a research tool to study the effects of (S)-2-HG in vivo.

The toxicological profile of TFMB-(S)-2-HG is evaluated in preclinical studies to ensure its safe use in research applications. As a prodrug of the oncometabolite (S)-2-hydroxyglutarate, the compound's toxicity is largely attributed to the effects of elevated (S)-2-HG levels in cells and tissues. (S)-2-HG is known to inhibit multiple dioxygenase enzymes, including TET2 and EglN prolyl hydroxylases, leading to epigenetic dysregulation and altered cellular metabolism. These effects can have significant toxicological consequences, particularly in highly proliferative tissues. In cell-based assays, TFMB-(S)-2-HG is typically used at concentrations ranging from 0.5 to 20 mM, and its cytotoxicity is assessed using standard viability assays such as MTT or CellTiter-Glo. The compound has been shown to inhibit the growth of various cancer cell lines, reflecting its potential anticancer activity, but it may also affect normal cells at high concentrations. In animal studies, the toxicity of TFMB-(S)-2-HG is evaluated by monitoring body weight, clinical observations, organ histopathology, hematological parameters, and clinical chemistry. High doses of the compound may lead to adverse effects related to the accumulation of (S)-2-HG, including alterations in DNA methylation, HIF signaling, and metabolic function. The compound's effects on osteogenic differentiation and Wnt/β-catenin signaling may also contribute to its toxicological profile, particularly in tissues where these pathways are critical for normal homeostasis. The toxicological evaluation of TFMB-(S)-2-HG is essential for determining the safe and effective dose range for in vivo studies. Researchers must carefully consider the potential off-target effects of (S)-2-HG accumulation when designing experiments and interpreting results. The compound is intended for research use only and is not approved for human therapeutic use. Standard safety precautions should be followed when handling TFMB-(S)-2-HG, including the use of appropriate personal protective equipment and adherence to institutional safety guidelines.

[1]. (R)-2-hydroxyglutarate is sufficient to promote leukemogenesis and its effects are reversible. Science. 2013;339(6127):1621-1625.

TFMB-(S)-2-HG is a research-grade compound exclusively for laboratory use. It is a trifluoromethylbenzyl (TFMB) ester prodrug of (S)-2-hydroxyglutarate [(S)-2-HG], a cell-membrane-permeant precursor used to probe (S)-2-HG enantiomer-specific cellular responses. The compound is widely used in cancer biology and epigenetics research to model IDH mutation-driven metabolic and transcriptional changes. It is a potent inhibitor of TET2 and EglN prolyl hydroxylases, and it downregulates Wnt3a and β-catenin protein expression. The compound also inhibits osteogenic differentiation of cells. TFMB-(S)-2-HG has potential for research into acute myeloid leukemia (AML) and other cancers where IDH mutations and 2-HG accumulation play a pathogenic role. The compound is not approved for human therapeutic use and is intended for research purposes only. It is available from various commercial suppliers as a high-purity reagent for biochemical and cell-based assays. The compound should be stored according to the manufacturer's recommendations, typically at -20°C or -80°C, to ensure stability. When handling TFMB-(S)-2-HG, researchers should follow standard safety protocols for handling chemical reagents, including the use of appropriate personal protective equipment and working in a well-ventilated area. The compound is soluble in DMSO and other organic solvents, and stock solutions should be prepared fresh or stored in aliquots to avoid repeated freeze-thaw cycles. TFMB-(S)-2-HG is a valuable tool for understanding the role of oncometabolites in cancer and other diseases, and it continues to be used in a wide range of research applications aimed at elucidating the mechanisms of IDH mutation-driven tumorigenesis and developing new therapeutic strategies.

C13H11F3O4

288.219254732132

288.06

1445703-64-9

TFMB-(R)-2-HG;1445700-01-5

99940754

White to light yellow solid powder

2.5

0

7

4

20

380

1

O1C(=O)CC[C@H]1C(OCC1=CC=CC(C(F)(F)F)=C1)=O

UXTLNUGKDZMPTC-JTQLQIEISA-N

InChI=1S/C13H11F3O4/c14-13(15,16)9-3-1-2-8(6-9)7-19-12(18)10-4-5-11(17)20-10/h1-3,6,10H,4-5,7H2/t10-/m0/s1

[3-(trifluoromethyl)phenyl]methyl (2S)-5-oxooxolane-2-carboxylate

TFMB(S)2HG; TFMB (S) 2 HG; TFMB-(S)-2-HG

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 : ~100 mg/mL (~346.96 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (8.67 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.

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