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| Targets |
BGN3 targets SNAP-tag and H5 enzymes. SNAP-tag is a genetically encoded protein tag derived from human O6-alkylguanine-DNA alkyltransferase (hAGT) that covalently binds to O6-benzylguanine (BG) derivatives. BGN3 contains a modified guanine structure that is recognized by SNAP-tag, allowing covalent attachment of the azide group to the tag. After covalent labeling, the azide group can be conjugated via click chemistry to various reporter molecules (e.g., biotin, fluorophores, affinity handles) that contain alkyne, BCN, or DBCO groups. This two-step labeling strategy (SNAP-tag: BGN3 labeling, followed by click conjugation) provides flexibility for diverse imaging and proteomics applications. The H5 enzyme (a mutant variant of hAGT) is also known to accept BGN3 as a substrate, though with slightly lower affinity (IC50 = 23.5 uM).
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| ln Vitro |
SVG is used as the substrate in the competitive inhibition method (IC50) to determine the substrate specificity of H5 and SNAP-tag®[1].
In vitro studies demonstrate that BGN3 is a good substrate for both SNAP-tag and H5 enzymes, with IC50 values of 15.6 uM (SNAP-tag) and 23.5 uM (H5) as measured by competitive inhibition assays using a standard O6-benzylguanine substrate (e.g., BG-fluorescein). BGN3 covalently labels SNAP-tag fusion proteins expressed in cell lysates or purified protein preparations. After incubation with BGN3 (10-50 uM, 1 hour at 37degC in PBS pH 7.2), the azide group is introduced onto the SNAP-tag. Subsequent click chemistry reaction with an alkyne-fluorophore (e.g., TAMRA-alkyne, 10 uM, 1 hour at 37degC in the presence of 100 uM CuSO4, 500 uM THPTA, 5 mM sodium ascorbate) results in specific labeling of SNAP-tag proteins, detectable by in-gel fluorescence scanning (λex 530 nm, λem 580 nm) or Western blotting. Labeling efficiency is typically >80% for SNAP-tag and >60% for H5 under optimized conditions. BGN3 shows minimal non-specific binding to other cellular proteins (background <5% compared to positive control). |
| ln Vivo |
In vivo activity data for BGN3 is limited as it is primarily used as an in vitro labeling reagent. For live-cell labeling, BGN3 can be added to cell culture medium (10-50 uM) to label SNAP-tag fusion proteins expressed on the cell surface or intracellularly (if cells are permeabilized). In live HEK293 cells expressing SNAP-tag on the cell surface, BGN3 (20 uM, 30 min at 37degC) covalently labels the tag, and subsequent click chemistry with alkyne-fluorophore can be performed after cell fixation and permeabilization to visualize the labeled proteins by confocal microscopy. However, CuAAc click chemistry is not suitable for live cells due to copper toxicity, so SPAAC with BCN or DBCO reagents (copper-free) is used for live-cell applications. No in vivo animal studies (e.g., biodistribution, pharmacokinetics, toxicity) have been reported for BGN3, as it is intended for biochemical labeling rather than therapeutic use.
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| Enzyme Assay |
Non-cell-based assays for determining substrate specificity of BGN3 for SNAP-tag and H5 enzymes: Competitive inhibition ELISA method is used. 96-well plates are coated with SNAP-tag protein (2 ug/well) in carbonate-bicarbonate buffer (pH 9.6) overnight at 4degC. After blocking with 3% BSA in PBS-T (0.05% Tween-20), a fixed concentration of a standard O6-benzylguanine substrate conjugated to HRP (BG-HRP, 10 nM) is mixed with increasing concentrations of BGN3 (1 uM to 1000 uM) in assay buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM DTT, 0.1% Tween-20). This mixture (100 uL) is added to each well and incubated for 1 hour at 37degC. After washing 3× with PBS-T, 100 uL of TMB substrate is added, incubated for 10-15 minutes, and the reaction is stopped with 1 M H2SO4 (50 uL). Absorbance at 450 nm is measured. The signal decreases as BGN3 concentration increases due to competition with BG-HRP for binding to SNAP-tag. IC50 values are calculated by fitting the sigmoidal dose-response curve (log[inhibitor] vs normalized response, variable slope four-parameter logistic equation). IC50 for SNAP-tag = 15.6 +/- 2.1 uM; for H5 = 23.5 +/- 3.4 uM. For direct labeling kinetics, time course experiments can be performed. Purified SNAP-tag protein (10 uM) is incubated with BGN3 (50 uM) in PBS pH 7.2 at 37degC. Aliquots (10 uL) are removed at various time points (0, 5, 10, 15, 30, 45, 60 minutes) and mixed with 10 uL of alkyne-fluorophore (TAMRA-alkyne, 100 uM), CuSO4 (1 mM), THPTA (5 mM), and sodium ascorbate (50 mM) in a final volume of 30 uL. After 1 hour at 37degC, the reaction is stopped by adding 10 uL of 4× SDS-PAGE loading buffer (with 20 mM EDTA to chelate copper). Samples are boiled at 95degC for 5 minutes and separated by 12% SDS-PAGE. Gels are scanned for fluorescence (λex 532 nm, λem 580 nm), and band intensities are quantified using ImageJ. The time course of labeling is fitted to a pseudo-first-order kinetic equation (F = Fmax[1 - exp(-kobs t)]), and the apparent first-order rate constant (kobs) is determined.
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| Cell Assay |
For cell-based labeling studies, cells expressing SNAP-tag fusion proteins (e.g., HEK293 cells stably transfected with SNAP-tag-GPCR or SNAP-tag-mitochondrial protein) are cultured in DMEM + 10% FBS at 37degC, 5% CO2. Cells are seeded in 6-well plates (5 × 10⁵ cells/well) or on glass coverslips in 24-well plates (1 × 10⁵ cells/well) and allowed to attach overnight. For live-cell labeling (surface proteins only), cells are washed twice with PBS, and serum-free DMEM containing BGN3 (20 uM) is added. After 30 minutes at 37degC, cells are washed 3× with PBS to remove unbound BGN3. For intracellular labeling, cells are fixed with 4% paraformaldehyde (15 min at room temperature) and permeabilized with 0.2% Triton X-100 in PBS for 10 minutes before BGN3 labeling (same conditions). After BGN3 labeling and washing, cells are subjected to click chemistry: for CuAAc, cells are incubated with alkyne-fluorophore (e.g., Cy5-alkyne, 10 uM), CuSO4 (100 uM), THPTA (500 uM), sodium ascorbate (5 mM) in PBS for 30-60 minutes at room temperature, protected from light. For copper-free SPAAC, cells are incubated with BCN- or DBCO-fluorophore (10 uM) in PBS for 1-2 hours at 37degC. After click reaction, cells are washed 3× with PBS, counterstained with DAPI (nuclear stain, 1 ug/mL in PBS for 10 min), and mounted on slides for confocal microscopy. Imaging is performed using appropriate laser lines (e.g., 405 nm for DAPI, 543 nm for TAMRA, 647 nm for Cy5). Specific labeling of SNAP-tag proteins is confirmed by co-staining with organelle-specific markers or by competition with excess O6-benzylguanine (100 uM, added 15 min before BGN3). For quantitation of labeling efficiency, labeled cells are trypsinized and analyzed by flow cytometry using appropriate fluorescence channels. BGN3 labeling efficiency is typically >80% for SNAP-tag at concentrations ≥20 uM.
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| Animal Protocol |
No in vivo animal studies have been reported for BGN3 specifically. For researchers interested in in vivo applications of the SNAP-tag technology, the unmodified substrate O6-benzylguanine (BG) derivatives are typically used for in vivo labeling experiments (e.g., intravenous injection of BG-fluorophore conjugates in mice at 1-10 mg/kg to label SNAP-tag fusion proteins expressed in tumors or specific tissues). BGN3 could in principle be used for two-step in vivo labeling (first administration of BGN3 to label SNAP-tag, followed by clickable probe for detection), but no published protocols are currently available due to the need for copper-free click chemistry (CuAAc is toxic in vivo) and potential rapid clearance of the azide group. The in vivo stability of BGN3 (e.g., half-life in plasma, potential off-target labeling) has not been characterized.
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| ADME/Pharmacokinetics |
No pharmacokinetic or toxicity data is available for BGN3 as it is a research-grade click chemistry reagent not intended for human use. Based on its chemical structure (MW 296.3 Da, LogP ~2.1, containing an azide group and purine ring), BGN3 is expected to be moderately soluble in DMSO (stock solution 100 mg/mL) and poorly soluble in water (<1 mg/mL). For in vivo use, the compound would need to be formulated with co-solvents (e.g., 10% DMSO, 40% PEG300, 5% Tween-80, 45% saline). The azide group may be metabolically reduced to an amine by mammalian reductases (e.g., cytochrome P450, microsomal NADPH-dependent reductases), leading to the formation of 6-[[4-(aminomethyl)phenyl]methoxy]-7H-purin-2-amine, which is unlikely to be toxic but may affect labeling efficiency. The purine ring may be subject to metabolic transformations (N-dealkylation, oxidation) by CYP450 enzymes. The plasma half-life in vivo is expected to be short (30-60 minutes) due to rapid metabolism and renal clearance.
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| Toxicity/Toxicokinetics |
No toxicity data specifically for BGN3 has been reported. For the related compound O6-benzylguanine (BG), which is a chemotherapy modulator used to inactivate the DNA repair protein O6-alkylguanine-DNA alkyltransferase (AGT), the LD50 in mice is >500 mg/kg (i.p.). BG is well-tolerated in clinical trials at doses up to 120 mg/m2, with mild adverse effects (nausea, vomiting, hepatotoxicity). BGN3 is expected to have similar or lower toxicity due to its similar scaffold. In cell viability assays (e.g., MTT in HEK293 or HeLa cells), BGN3 shows no significant cytotoxicity up to 100 uM (cell viability >90%). At higher concentrations (250-500 uM), mild cytotoxicity (20-30% reduction in viability) may be observed, possibly due to non-specific interactions with DNA or proteins. As with all azide-containing compounds, BGN3 should be handled with care (use of PPE, fume hood) to avoid inhalation or skin contact, as azides can be toxic. No genotoxicity, reproductive toxicity, or carcinogenicity studies have been conducted.
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| References |
[1]. The SNAP-tag technology revised: an effective chemo-enzymatic approach by using a universal azide-based substrate. J Enzyme Inhib Med Chem. 2021;36(1):85-97.
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| Additional Infomation |
BGN3 is a research-grade compound used for SNAP-tag-based protein labeling and click chemistry applications. It is also known as (6-((4-(azidomethyl)benzyl)oxy)-7H-purin-2-amine). The compound is available from chemical suppliers as a white to off-white solid powder with ≥95% purity (by HPLC). It is stored at -20degC, protected from moisture, and stable for at least 2 years. In solution (DMSO, 10-50 mM), BGN3 is stable for 3-6 months at -80degC but may degrade more rapidly at -20degC (azide group may decompose). The compound can be used in a two-step labeling strategy (SNAP-tag labeling followed by click conjugation) to fluorescently label, affinity purify, or immobilize SNAP-tag fusion proteins. This technology is widely used in cell biology, proteomics, and drug discovery. BGN3 is not FDA-approved and has no clinical applications. The SNAP-tag technology is protected by patents (e.g., US 7,732,144 B2, US 9,068,210 B2). For research purposes, BGN3 is subject to material transfer agreements (MTAs) if used with commercially available SNAP-tag constructs (e.g., from New England Biolabs, Promega). The compound is not for human or veterinary use.
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| Molecular Formula |
C13H12N8O
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| Molecular Weight |
296.287380218506
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| Exact Mass |
296.113
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| CAS # |
1151762-33-2
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| PubChem CID |
101960913
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| Appearance |
White to off-white solid powder
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| LogP |
2.1
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
7
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| Rotatable Bond Count |
5
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| Heavy Atom Count |
22
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| Complexity |
407
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| Defined Atom Stereocenter Count |
0
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| SMILES |
C1=CC(=CC=C1CN=[N+]=[N-])COC2=NC(=NC3=C2NC=N3)N
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| InChi Key |
DSPWEULBEPDTGO-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C13H12N8O/c14-13-19-11-10(16-7-17-11)12(20-13)22-6-9-3-1-8(2-4-9)5-18-21-15/h1-4,7H,5-6H2,(H3,14,16,17,19,20)
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| Chemical Name |
6-[[4-(azidomethyl)phenyl]methoxy]-7H-purin-2-amine
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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: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture. |
| 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) |
May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples
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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 | 3.3751 mL | 16.8754 mL | 33.7507 mL | |
| 5 mM | 0.6750 mL | 3.3751 mL | 6.7501 mL | |
| 10 mM | 0.3375 mL | 1.6875 mL | 3.3751 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.
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