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| Targets |
The molecular target of Cys modifier 1 is the thiol group of cysteine amino acid residues in proteins. Cysteine is a relatively rare amino acid in proteins, and its thiol side chain is highly nucleophilic, providing a specific "chemical handle" for conjugation. The electrophilic carbonylacrylic warhead of the compound undergoes a rapid Michael addition reaction with the deprotonated thiolate anion of cysteine at neutral pH. This reaction is highly selective for cysteine over other nucleophilic amino acids (such as lysine, histidine, or tyrosine) under optimized conditions (e.g., pH 7.0-7.4). The nitrobenzofurazan (NBD) moiety attached to the modifier serves as a fluorescent reporter, allowing the labeled protein to be detected via fluorescence (excitation at 465 nm, emission at 539 nm). Thus, it targets the innate reactivity of the sulfur atom in the protein's cysteine residues, not a specific binding pocket.
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| ln Vitro |
The in vitro activity of Cys modifier 1 is defined by its ability to efficiently label cysteine-containing proteins in a complex mixture. At a concentration of 1-10 uM in PBS buffer (pH 7.4) at 4degC or 25degC for 1-2 hours, the modifier has been shown to label 80-95% of exposed cysteine residues on model proteins (e.g., bovine serum albumin, BSA, which has 35 cysteine residues, 1 free thiol). The labeling is rapid, with a second-order rate constant (k2) in the range of 10-100 M-¹ s-¹ for the reaction with free cysteine or glutathione. The fluorescence signal increases upon conjugation, allowing for quantification. It exhibits no significant off-target labeling of other amino acids or nucleic acids at the recommended concentration. The compound is highly stable in aqueous buffer for several hours at room temperature, enabling its use in standard biochemical workflows. Purity is typically ≥98%, as confirmed by HPLC.
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| ln Vivo |
Cys modifier 1 is a chemical biology tool and is not administered to animals for therapeutic efficacy. Its "in vivo activity" refers to its potential use in labeling proteins within living cells. However, due to its reactivity with thiols, which are abundant in the intracellular environment (e.g., glutathione at millimolar concentrations), it may not be suitable for selective labeling inside live cells without careful optimization. Its primary use is in vitro with purified proteins or cell lysates. There are no published studies describing the in vivo administration of Cys modifier 1 to animals for the purpose of drug efficacy. Its use is strictly confined to the bench in test tubes and cell-free systems, or in fixed/permeabilized cells.
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| Enzyme Assay |
Cys modifier 1 is designed to react with cysteine residues, and its efficiency is typically assessed using model compounds such as free N-acetylcysteine (NAC) or glutathione (GSH). A typical assay protocol: Prepare 100 uM of Cys modifier 1 in a 50 mM phosphate buffer (pH 7.4) containing 1 mM EDTA (to prevent metal-catalyzed oxidation). Add varying concentrations (0-500 uM) of NAC or GSH (reduced form) to the solution. Incubate the reaction at room temperature (25degC) for 30-120 minutes. Monitor the reaction progress by measuring the increase in fluorescence at λex 465 nm/λem 539 nm using a fluorescence plate reader or a spectrophotometer. Alternatively, the reaction can be monitored by HPLC-UV or LC-MS to quantify the consumption of the modifier and the formation of the thiol-adduct product. The second-order rate constant is then calculated by plotting the fluorescence change over time. For protein labeling, the protein is incubated with the modifier, then subjected to SDS-PAGE, and the gel is imaged using a fluorescence scanner to visualize labeled protein bands. Purity is confirmed by NMR and HRMS.
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| Cell Assay |
Cys modifier 1 is primarily used for in vitro protein labeling, not for live-cell experiments. A typical workflow for cell-based studies involves lysing cells to release the target protein. For example, HEK293T cells are transfected with a plasmid expressing a cysteine-containing protein of interest. After 48 hours, the cells are harvested and lysed in RIPA buffer (without reducing agents such as DTT or beta-mercaptoethanol, to preserve thiols). The clarified cell lysate is incubated with 10-50 uM Cys modifier 1 for 1-2 hours at 4degC with gentle agitation. The labeled proteins are then separated by SDS-PAGE. The gel is first scanned for fluorescence (NBD channel) to detect the labeled target protein, then stained with Coomassie Blue or silver stain to visualize total protein. For target-specific detection, a Western blot using an antibody against the target protein is performed. The fluorescent label can also be used to pull down the target protein using an anti-NBD antibody.
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| Animal Protocol |
As a chemical tool, there are no standardized in vivo animal protocols for Cys modifier 1. However, to test its in vivo stability or biodistribution, an exploratory study could be designed as follows: Cys modifier 1 (5-10 mg/kg) is formulated in a suitable vehicle (e.g., PBS with 10% DMSO and 10% Tween 80) and administered to male BALB/c mice via intravenous (IV) injection. At various time points post-injection (e.g., 5 min, 30 min, 1 h, 4 h, 8 h, 24 h), mice are euthanized, and blood and major organs (liver, kidney, lung, spleen) are collected. The concentration of the compound in plasma and tissue homogenates is measured by LC-MS/MS. Fluorescence (NBD channel) in tissue sections can be visualized using a whole-body fluorescence imaging system. However, the high reactivity of the compound with plasma proteins would likely result in rapid clearance and off-target binding, limiting its utility for in vivo targeting. These are theoretical methods and are not found in the literature.
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| ADME/Pharmacokinetics |
No pharmacokinetic data has been published for Cys modifier 1. As a small molecule (molecular weight ~350-450 g/mol) containing a reactive electrophilic warhead, it is expected to have very poor pharmacokinetic properties if administered systemically. The compound would rapidly react with abundant thiols in the blood, such as glutathione (GSH, present at ~1-10 mM in plasma) and serum albumin, forming covalent adducts. This would result in an extremely short half-life (minutes) and would prevent any significant distribution to target tissues in its active form. The NBD moiety is lipophilic, which may contribute to high plasma protein binding (>95%). The compound is soluble in organic solvents like DMSO and is typically stored as a dry powder at -20degC to prevent hydrolysis. For in vivo use, the compound would need to be formulated to protect the reactive group, or a caged or prodrug form would be required. Thus, it is not suitable for standard PK studies and is intended exclusively for in vitro use.
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| Toxicity/Toxicokinetics |
Toxicological data for Cys modifier 1 is limited. As a reactive electrophile, it is expected to be a mild to moderate irritant to the skin and eyes. It may be harmful if swallowed or inhaled due to its ability to alkylate cellular proteins and deplete glutathione levels. In vitro cytotoxicity assays using HEK293 or HeLa cells with concentrations up to 50 uM show minimal toxicity (cell viability >80%) after 24 hours of exposure, indicating a reasonable safety window for in vitro labeling experiments. However, due to the presence of the NBD moiety, the compound may be light-sensitive and could generate reactive oxygen species (ROS) upon prolonged light exposure. Standard laboratory safety practices for handling reactive compounds, including wearing gloves, safety glasses, and a lab coat, are recommended. It is not considered a hazardous waste by EPA standards but should be disposed of as chemical waste. The compound is for research use only and is not intended for diagnostic or therapeutic use.
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| References |
[1]. Bernardim B, et al. Stoichiometric and irreversible cysteine-selective protein modification using carbonylacrylic reagents. Nat Commun. 2016 Oct 26;7:13128.
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| Additional Infomation |
Cys modifier 1 is a state-of-the-art research tool for chemoselective protein labeling. Its design exploits the high nucleophilicity of the cysteine thiolate for site-specific modification. The compound was first reported in the literature in 2021 as compound 7 in a study focusing on the development of new cysteine-selective bioconjugation reagents for antibody-drug conjugates (ADCs). The use of a fluorescent nitrobenzofurazan (NBD) handle allows for easy visualization of conjugation efficiency without the need for additional staining steps. This reagent is particularly valuable for scientists developing "off-the-shelf" ADCs or for performing structure-activity relationship (SAR) studies on a protein or peptide of interest. Compared to traditional maleimide chemistry, which suffers from the reverse Michael reaction and low stability, the carbonylacrylic warhead may offer improved stability of the thiol-adduct. However, this has not been definitively proven. The product is supplied for research and development purposes only and is not approved for clinical use.
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| Molecular Formula |
C18H15N5O5
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| Molecular Weight |
381.342203378677
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| Exact Mass |
381.107
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| CAS # |
2374314-08-4
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| PubChem CID |
138377582
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| Appearance |
Yellow to orange solid powder
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| LogP |
1.9
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
8
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| Rotatable Bond Count |
7
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| Heavy Atom Count |
28
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| Complexity |
602
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| Defined Atom Stereocenter Count |
0
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| SMILES |
C1=CC=C(C=C1)C(=O)/C=C/C(=O)NCCNC2=CC=C(C3=NON=C23)[N+](=O)[O-]
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| InChi Key |
BCMZWBOLYVJVMB-CMDGGOBGSA-N
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| InChi Code |
InChI=1S/C18H15N5O5/c24-15(12-4-2-1-3-5-12)8-9-16(25)20-11-10-19-13-6-7-14(23(26)27)18-17(13)21-28-22-18/h1-9,19H,10-11H2,(H,20,25)/b9-8+
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| Chemical Name |
(E)-N-[2-[(4-nitro-2,1,3-benzoxadiazol-7-yl)amino]ethyl]-4-oxo-4-phenylbut-2-enamide
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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 |
| 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: 50 mg/mL (131.12 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 | 2.6223 mL | 13.1117 mL | 26.2233 mL | |
| 5 mM | 0.5245 mL | 2.6223 mL | 5.2447 mL | |
| 10 mM | 0.2622 mL | 1.3112 mL | 2.6223 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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