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25mg |
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Purity: ≥98%
Targets |
Fluorescent dye; HMBR targets the Y-FAST protein tag (KD = 0.13 ± 0.01 μM; KON = 6.3 ± 0.9 × 107 M−1·s−1, KOFF = 6.3 ± 0.7 s−1 at pH 7.4 and 25°C).[1]
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ln Vitro |
HMBR exhibits minimal fluorescence in free form (quantum yield Φ = 0.04% at pH 5.8 and 0.2% at pH 10.5) but upon binding to Y-FAST, fluorescence quantum yield increases to 33% with maximal emission at 540 nm, and absorption shifts from 401 nm (free) to 481 nm (bound), indicating deprotonation-induced activation; this dual spectroscopic change ensures high signal-to-noise ratios in cellular imaging due to negligible background from unbound HMBR.
In mammalian cells (e.g., HeLa, HEK293), HMBR (5–10 μM) rapidly labels Y-FAST fusion proteins in various subcellular compartments (cytoplasm, nucleus, membrane, mitochondria, Golgi, cytoskeleton) within 10 seconds, as confirmed by confocal microscopy; brightness and photostability are comparable to EGFP and UnaG, with no cytotoxicity observed at imaging concentrations. Flow cytometry and microscopy in bacteria (E. coli), yeast (S. cerevisiae), and mammalian cells confirm HMBR is cell-permeant, generates negligible nonspecific fluorescence, and does not bind wild-type PYP, demonstrating high labeling specificity for Y-FAST.[1] HMBR (20 μM) rapidly activates green fluorescence (λem = 541 nm) in *E. coli* Nissle 1917 expressing FAST (EcN-FAST) within minutes; fluorescence intensity is directly proportional to bacterial CFUs (R2 = 0.999), enabling quantitative viability assessment via microplate reader or flow cytometry. Reversible fluorescence switching: Pre-activated EcN-FAST (green) fully transitions to red emission within minutes upon addition of HBR-3,5-DOM (red fluorogen), confirmed by LSCM imaging and fluorescence microplate quantification; dynamic exchange eliminates spectral crosstalk with GFP/mCherry-expressing bacteria. Fluorescence turn-off: Repeated PBS washing dissociates bound HMBR, reducing signal to baseline within 5 washes (validated by LSCM, flow cytometry, and microplate reader).[2] |
ln Vivo |
In zebrafish embryos, HMBR (5–10 μM) permeates tissues and specifically labels Y-FAST fusion proteins coexpressed with mCherry within 20–30 minutes, showing identical expression patterns to mCherry controls; spinning-disk confocal microscopy confirms in vivo specificity and absence of labeling in PYP-expressing embryos.
Prolonged exposure (19 hours) to HMBR causes no mortality or developmental anomalies in zebrafish embryos, indicating low toxicity and suitability for long-term developmental imaging. Real-time monitoring in cell-free expression systems shows HMBR-Y-FAST fluorescence appears within 10 minutes of protein synthesis initiation, outperforming mCherry (50-minute delay) and matching Firefly luciferase kinetics, enabling near-real-time reporting of rapid processes like protein synthesis.[1] In ICR mice gut microbiota, oral HMBR (5 mM, 100 μL) activates EcN-FAST fluorescence within 1 hour; peak colonization occurs at 8 hours (IVIS imaging), with signal distribution along the intestinal tract. In 4T1 tumor-bearing mice, intratumoral HMBR injection generates sustained fluorescence for 24+ hours due to nutrient-rich microenvironment; emission switches from green to red upon HBR-3,5-DOM injection, enabling dual-modal imaging in hypoxic tissues. Linear correlation (R2 > 0.98) between IVIS fluorescence intensity and CFU counts in gut contents confirms HMBR's utility for real-time bacterial vitality quantification in vivo.[2] |
Enzyme Assay |
Binding kinetics were quantified using stopped-flow experiments: HMBR and Y-FAST were mixed in pH 7.4 buffer at 25°C, and fluorescence increase was monitored to determine on-rate (KON) and off-rate (KOFF) constants; data fitting yielded KON = 6.3 ± 0.9 × 107 M−1·s−1 and KOFF = 6.3 ± 0.7 s−1.
Dissociation constants (KD) were measured via fluorescence titration: Serial dilutions of HMBR were added to purified Y-FAST, and fluorescence intensity (λex = 481 nm, λem = 540 nm) was plotted against concentration to calculate KD = 0.13 ± 0.01 μM using nonlinear regression analysis. Fluorogen activation specificity was assessed by comparing absorption and emission spectra of free vs. bound HMBR in buffered solutions, confirming the red shift and quantum yield increase are exclusive to Y-FAST binding.[1] |
Animal Protocol |
For zebrafish embryo labeling: Wild-type (Ab strain) embryos at one-cell stage were injected with Y-FAST mRNA and incubated at 28°C in Volvic mineral water; HMBR was dissolved in culture medium to 10 μM and added at 50% epiboly stage, with embryos imaged at 24 hours post-fertilization using spinning-disk confocal microscopy.
Reversibility studies: Embryos expressing Y-FAST were incubated with 10 μM HMBR for 20 minutes, washed twice (20 minutes each) in fluorogen-free medium, and reincubated with HMBR; fluorescence was monitored before, during, and after washes to confirm rapid labeling/unlabeling cycles. Toxicity assessment: Groups of ~50 embryos were exposed to HMBR (concentrations up to 10 μM) from 50% epiboly to 24 hours post-fertilization; viability and development were evaluated for mortality/morphological anomalies.[1] Gut imaging: Female ICR mice orally gavaged with 1×108 CFU EcN-FAST, followed by 100 μL HMBR (5 mM in water) 1 hour later; euthanized at 1/2/4/8/24 h for intestinal tract IVIS imaging. Tumor imaging: 4T1 tumor-bearing BALB/c mice received intratumoral injection of 1×107 CFU EcN-FAST, followed by 50 μL HMBR after 1 h; imaged at 0/3/6/24 h post-injection. Reversibility studies: Mice with HMBR-activated EcN-FAST orally/injection-administered HBR-3,5-DOM; GI tract/tumors harvested at 0.5/1/2/4 h for dual-channel IVIS analysis.[2] |
ADME/Pharmacokinetics |
Cell permeability assays in HeLa and HEK293 cells show HMBR rapidly enters cells (≤10 seconds), with intracellular concentrations reflecting extracellular levels, as confirmed by titration matching in vitro KD values.
In zebrafish embryos, HMBR achieves tissue-wide distribution within 20–30 minutes, with washout kinetics indicating reversible binding and rapid clearance; no data on metabolism, excretion, or bioavailability are reported.[1] |
Toxicity/Toxicokinetics |
Cytotoxicity assays in mammalian cells (HeLa, HEK293) reveal no adverse effects at HMBR concentrations ≤10 μM after prolonged exposure, as assessed by cell viability and morphology.
In zebrafish embryos, no lethality or developmental defects occur after 19-hour exposure to 10 μM HMBR, supporting low in vivo toxicity; plasma protein binding and drug interactions were not evaluated.[1] |
References |
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Additional Infomation |
HMBR (4-hydroxy-3-methylbenzylidene-rhodamine) is a fluorogenic ligand designed for reversible, dynamic binding to Y-FAST, enabling tunable fluorescence activation via deprotonation and immobilization; it facilitates multiplexed imaging through sequential labeling/unlabeling in live cells.
Mechanism: Binding to Y-FAST stabilizes the deprotonated form of HMBR (pKA = 8.7 ± 0.1), causing a 80-nm absorption red shift and >100-fold fluorescence increase, which is instantaneous and reversible. Applications: Used for real-time protein tracking, super-resolution microscopy (due to blinking from dynamic binding), and FRET-based biosensors; no FDA approvals or warnings are noted, as it is a research tool.[1] HMBR enables oxygen-independent fluorescence activation, critical for imaging anaerobic environments (gut/tumors); its reversible binding allows dynamic on/off switching and emission band swapping (green→red) via fluorogen exchange. Mechanism: Binding to FAST stabilizes deprotonated HMBR, inducing 80-nm absorption red shift and >100-fold quantum yield increase (Φ = 33%). Applications: Serves as a "smart tag" for living bacterial probes (e.g., EcN-FAST) to track microbiota distribution, colonization dynamics, and viability in mammalian hosts; no cytotoxicity observed at 20 μM in vitro or 5 mM in vivo.[2] |
Molecular Formula |
C11H9NO2S2
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Molecular Weight |
251.32466006279
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Exact Mass |
251.007
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CAS # |
1287651-36-8
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PubChem CID |
118628175
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Appearance |
Yellow to orange solid powder
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LogP |
2.8
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Hydrogen Bond Donor Count |
2
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Hydrogen Bond Acceptor Count |
4
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Rotatable Bond Count |
1
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Heavy Atom Count |
16
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Complexity |
354
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Defined Atom Stereocenter Count |
0
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SMILES |
S1/C(=C\C2=CC=C(O)C(C)=C2)/C(=O)NC1=S
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InChi Key |
WUYJNCRVBZWAOK-UITAMQMPSA-N
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InChi Code |
InChI=1S/C11H9NO2S2/c1-6-4-7(2-3-8(6)13)5-9-10(14)12-11(15)16-9/h2-5,13H,1H3,(H,12,14,15)/b9-5-
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Chemical Name |
(5Z)-5-[(4-hydroxy-3-methylphenyl)methylidene]-2-sulfanylidene-1,3-thiazolidin-4-one
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Synonyms |
HMBR; 1287651-36-8; SCHEMBL17397853; (5Z)-5-[(4-hydroxy-3-methylphenyl)methylidene]-2-sulfanylidene-1,3-thiazolidin-4-one;
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HS Tariff Code |
2934.99.9001
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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)
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Solubility (In Vitro) |
DMSO: 125 mg/mL (497.37 mM)
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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
Injection Formulation 1: DMSO : Tween 80: Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *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). View More
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)] 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). View More
Oral Formulation 3: Dissolved in PEG400  (Please use freshly prepared in vivo formulations for optimal results.) |
Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
1 mM | 3.9790 mL | 19.8950 mL | 39.7899 mL | |
5 mM | 0.7958 mL | 3.9790 mL | 7.9580 mL | |
10 mM | 0.3979 mL | 1.9895 mL | 3.9790 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.
Calculation results
Working concentration: mg/mL;
Method for preparing DMSO stock solution: mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation::Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O,mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.