69-78-3

Basic information

• Product Name: 3-Carboxy-4-nitrophenyl disulfide
• Synonyms: DTNB;ellmann's reagent;ELLMAN'S REAGENT;ELLMANS' REAGENT;BIS(3-CARBOXY-4-NITROPHENYL) DISULFIDE;3-CARBOXY-4-NITROPHENYL DISULFIDE;5,5'-DITHIOBIS(2-NITROBENZOIC ACID);5,5-DITHIO-BIS(2-NITROBENZOIC ACID)
• CAS NO: 69-78-3
• Molecular Formula: C₁₄H₈N₂O₈S₂
• Molecular Weight: 396.35
• EINECS: 200-714-4
• Product Categories: Phenyls & Phenyl-Het;Phenyls & Phenyl-Het;marker
• Mol File: 69-78-3.mol
• Article Data: 19

Chemical Properties

• Melting Point: 240-245 °C (dec.)(lit.)
• Boiling Point: 671.863 °C at 760 mmHg
• Flash Point: 360.13 °C
• Appearance: /
• Density: 1.67
• Vapor Pressure: 5.73E-19mmHg at 25°C
• Refractive Index: 1.6900 (estimate)
• Storage Temp.: Store at RT.
• Solubility: Do you have solubility information on this product that you woul
• PKA: 1.57±0.25(Predicted)
• Water Solubility: Soluble in water and ethanol.
• Stability: Stable. Incompatible with strong bases, strong oxidizing agents.
• Merck: 14,3551
• BRN: 1896821
• CAS DataBase Reference: 3-Carboxy-4-nitrophenyl disulfide(CAS DataBase Reference)
• NIST Chemistry Reference: 3-Carboxy-4-nitrophenyl disulfide(69-78-3)
• EPA Substance Registry System: 3-Carboxy-4-nitrophenyl disulfide(69-78-3)

Safety Data

• Hazard Codes: Xi
• Statements: 36/37/38
• Safety Statements: 26-36
• WGK Germany: 3
• RTECS: DG9650000
• F: 10
• TSCA: Yes
• Hazardous Substances Data: 69-78-3(Hazardous Substances Data)

Usage

Chemical Properties

Crystalline

Uses

Different sources of media describe the Uses of 69-78-3 differently. You can refer to the following data:
1. 5,5′-Dithiobis(2-nitrobenzoic acid) is an membrane-impermeant thiol/disulfide exchange blocker. It is also used to quantify the number or concentration of thiol groups in a sample.
2. Reagent for determination of sufhydryl groups.5,5'-dithiobis-(2-nitrobenzoic acid) is widely utilized as a non-fluorescent probe to quantify the number or the concentration of thiol groups in a sample. It acts as a cleavable cross-linking agent. It finds an important application as a sensitive reagent for determining free sulfhydryl content in proteins, peptides, and tissues. It is also employed in high-performance liquid chromatography (HPLC) to determine the alkylthiol content.
3. 5,5′-Dithiobis(2-nitrobenzoic acid) is a water soluble reagent mainly used for the derivatization of sulfhydryl groups.

Definition

ChEBI: An organic disulfide that results from the formal oxidative dimerisation of 2-nitro-5-thiobenzoic acid. An indicator used to quantify the number or concentration of thiol groups.

General Description

5,5′-Dithiobis(2-nitrobenzoic acid) is also called DTNB or Ellman′s reagent. It is a water soluble reagent, which interacts with thiol groups, and can be used conveniently at neutral pH. Thiolate anion (RS-) reacts with Ellman′s reagent (DTNB2-), to produce 2-nitro-5-thiobenzoate anion (TNB2-) and one mixed disulfide (R-S-TNB-). TNB2- has a high molar absorption coefficient, and absorbs at 412nm. This absorption is independent of pH, at pH> 7.3.

InChI:InChI=1/C14H8N2O8S2/c17-13(18)9-5-7(1-3-11(9)15(21)22)25-26-8-2-4-12(16(23)24)10(6-8)14(19)20/h1-6H,(H,17,18)(H,19,20)/p-2

Synthetic route

5-bromo-2-nitrobenzoic acid (6950-43-2) → 5,5'-dithiobis-(2-nitrobenzoic acid) (69-78-3)

Conditions	Yield
With sodium sulfide In water at 50℃; for 2h; pH=> 3.5;	72.3%

5-chloro-2-nitrobenzoic acid (2516-95-2) → 5,5'-dithiobis-(2-nitrobenzoic acid) (69-78-3)

Conditions	Yield
With sodium sulfide; sodium hydroxide anschliessendes Behandeln mit Jod und KI;

tert.-butylhydroperoxide (75-91-2) + 5-thio-2-nitrobenzoic acid (15139-21-6) → 5,5'-dithiobis-(2-nitrobenzoic acid) (69-78-3) + tert-butyl alcohol (75-65-0)

Conditions	Yield
With ethylenediaminetetraacetic acid; MES buffer; selenosubtilisin (ESeSAr form) at 25℃; Rate constant; pH 5.5; other seleno- reagent, (Km)t-BuOOH;

5-thio-2-nitrobenzoic acid (15139-21-6) → 5,5'-dithiobis-(2-nitrobenzoic acid) (69-78-3)

Conditions	Yield
With potassium hydroxide; dihydrogen peroxide In water
With dithionite(2-); cetyltrimethylammonim bromide In water Equilibrium constant;
With NaOH buffer; 1-phenylethyl hydroperoxide; seleno-subtilisin Carlsberg; citric acid at 20℃; pH=5.5; Enzyme kinetics; Oxidation;

3-carboxylate-4-nitrothiophenoxide ion (77874-90-9) → 5,5'-dithiobis-(2-nitrobenzoic acid) (69-78-3)

Conditions	Yield
With 1-oxido-1,2-benziodoxol-3(1H)-one In water at 25℃; Kinetics; Rate constant; Mechanism; pH=8, μ=00.1(KCl), the reagent and the title compound were microencapsulated in vesicles of dioctadecyldimethylammonium chloride;

Cumene hydroperoxide (80-15-9) + 5-thio-2-nitrobenzoic acid (15139-21-6) → 5,5'-dithiobis-(2-nitrobenzoic acid) (69-78-3) + 1-methyl-1-phenylethyl alcohol (617-94-7)

Conditions	Yield
Stage #1: 5-thio-2-nitrobenzoic acid With supramolecular artificial glutathione peroxidase (SGPxmax) In aq. phosphate buffer at 36℃; for 0.05h; pH=7; Enzymatic reaction; Stage #2: Cumene hydroperoxide In aq. phosphate buffer Kinetics; Catalytic behavior; Reagent/catalyst;
With C17H28O3Te In aq. phosphate buffer; ethanol at 25℃; pH=7; Kinetics; Solvent;

C18H27NO5SSe → 5,5'-dithiobis-(2-nitrobenzoic acid) (69-78-3) + bis(11-hydroxyundecyl) diselenide (1204351-95-0)

Conditions	Yield
Irradiation;

meta-bromotoluene (591-17-3) → 5,5'-dithiobis-(2-nitrobenzoic acid) (69-78-3)

Conditions	Yield
Multi-step reaction with 3 steps 1: nitric acid; sulfuric acid / 2 h / 45 - 55 °C 2: potassium permanganate / water / 50 °C 3: sodium sulfide / water / 2 h / 50 °C / pH > 3.5 View Scheme

5,5'-dithiobis-(2-nitrobenzoic acid) (69-78-3) + MGSSHHHHHHSSGLVPRGSASQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGR TLSDYNIQKESTLHLVLRLRGC disulfide → MGSSHHHHHHSSGLVPRGSASQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGR TLSDYNIQKESTLHLVLRLRGC* (C* = Cys-SS-3-carboxy-4-nitrophenyl)

Conditions	Yield
Stage #1: MGSSHHHHHHSSGLVPRGSASQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGR TLSDYNIQKESTLHLVLRLRGC disulfide With D,L-dithiothreitol In aq. phosphate buffer at 37℃; for 0.333333h; pH=7.4; Stage #2: 5,5'-dithiobis-(2-nitrobenzoic acid) In aq. phosphate buffer at 20℃; for 0.0333333h; pH=7.4;	100%

5,5'-dithiobis-(2-nitrobenzoic acid) (69-78-3) → 5-thio-2-nitrobenzoic acid (15139-21-6)

Conditions	Yield
With sodium tetrahydroborate In ethanol; water at 0 - 20℃;	97%
With dithionite(2-); cetyltrimethylammonim bromide In water Kinetics; Equilibrium constant; further reagents, catalysts;
With 2-(N,N-dimethylamino)ethanol; 2-mercaptoethylamine hydrochloride In water at 25℃; for 1h; Yield given;

1-hydroxy-pyrrolidine-2,5-dione (6066-82-6) + 5,5'-dithiobis-(2-nitrobenzoic acid) (69-78-3) → 5,5'-dithiobis(2-nitrobenzoic acid succinimidyl diester) (60129-38-6)

Conditions	Yield
With dicyclohexyl-carbodiimide In tetrahydrofuran at 25 - 28℃; for 2h;	97%
With dicyclohexyl-carbodiimide In dichloromethane; N,N-dimethyl-formamide at 4℃;
With dicyclohexyl-carbodiimide In tetrahydrofuran at 25℃; for 12h;

NaHCO + 5,5'-dithiobis-(2-nitrobenzoic acid) (69-78-3) → methyl 2-(2-methoxycarbonyl-3-nitrophenyl)disulfanyl-6-nitrobenzoate (115215-73-1)

Conditions	Yield
With pyridine; thionyl chloride; citric acid In methanol; dichloromethane; N,N-dimethyl-formamide	92%

4-hydroxy-1-methyl-2(1H)-quinolone (1677-46-9) + 5,5'-dithiobis-(2-nitrobenzoic acid) (69-78-3) → 3-(3-carboxy-4-nitrophenylthio)-4-hydroxy-1-methyl-2(1H)-quinolone

Conditions	Yield
With potassium carbonate In N,N-dimethyl-formamide at 90 - 95℃; for 5h; Substitution;	86%

5,5'-dithiobis-(2-nitrobenzoic acid) (69-78-3) + [Na(18-crown-6)]2[Fe2(μ-S)2(NO)4] → [Na(18-crown-6)][Fe(SC6H3(NO2)COOH)2(NO)2] (1335311-62-0)

Conditions	Yield
In acetonitrile byproducts: S8; (N2); stirring soln. of iron compd. and disulfide deriv. in CH3CN at room temp. for 30 min; evapn., dissolving in THF/diethyl ether (1:1), filtration through celite, addn. of hexane, isolation of ppt., elem. anal.;	85%

5,5'-dithiobis-(2-nitrobenzoic acid) (69-78-3) → 2-nitro-5-mercaptobenzyl alcohol (105728-34-5) + 5-(methylthio)-2-nitrobenzyl alcohol (105762-12-7)

Conditions	Yield
With dimethylsulfide borane complex In 1,2-dimethoxyethane for 3h; Heating;	A 80% B n/a

5,5'-dithiobis-(2-nitrobenzoic acid) (69-78-3) + 1-dodecylthiol (112-55-0) → 6-nitro-3-(dodecyldisulfamyl) benzoic acid

Conditions	Yield
In ethanol Ambient temperature;	75%

1-Bromo-11-hydroxyundecane (1611-56-9) + 5,5'-dithiobis-(2-nitrobenzoic acid) (69-78-3) → bis(11-hydroxyundecyl) 5,5'-dithiobis(2-nitrobenzoate)

Conditions	Yield
Stage #1: 5,5'-dithiobis-(2-nitrobenzoic acid) With sodium hydride In N,N-dimethyl-formamide; mineral oil at 60℃; for 0.25h; Inert atmosphere; Stage #2: 1-Bromo-11-hydroxyundecane In N,N-dimethyl-formamide; mineral oil at 60℃; Inert atmosphere;	75%

5,5'-dithiobis-(2-nitrobenzoic acid) (69-78-3) + thiophenol (108-98-5) → 2-nitro-5-(phenyldisulfaneyl)benzoic acid

Conditions	Yield
In ethanol; acetonitrile at 20℃;	75%

1,2,3-Benzotriazole (95-14-7) + 5,5'-dithiobis-(2-nitrobenzoic acid) (69-78-3) → {5-[3-(benzotriazole-1-carbonyl)-4-nitrophenyldisulfanyl]-2-nitrophenyl}benzotriazol-1-ylmethanone (1234804-46-6)

Conditions	Yield
Stage #1: 1,2,3-Benzotriazole With thionyl chloride In dichloromethane at 25℃; for 0.5h; Stage #2: 5,5'-dithiobis-(2-nitrobenzoic acid) In dichloromethane	74%

5,5'-dithiobis-(2-nitrobenzoic acid) (69-78-3) → 5-thio-2-nitrobenzoic acid

Conditions	Yield
2-(N,N-dimethylamino)ethanol	70%

5,5'-dithiobis-(2-nitrobenzoic acid) (69-78-3) + 1,8-diazabicyclo[5.4.0]undec-7-ene (6674-22-2) → C9H16N2*C14H8N2O8S2 (1379592-24-1)

Conditions	Yield
In chloroform	68%

5,5'-dithiobis-(2-nitrobenzoic acid) (69-78-3) + 7-tridecanethiol → 2-nitro-5-(tridecan-7-yldisulfaneyl)benzoic acid

Conditions	Yield
With trimethylamine In dichloromethane for 1h;	63%

1,2-bis(4'-pyridyl)ethane (4916-57-8) + 5,5'-dithiobis-(2-nitrobenzoic acid) (69-78-3) + water (7732-18-5) + manganese (II) acetate tetrahydrate (6156-78-1) → C14H6N2O8S2(2-)*Mn(2+)*3H2O*C12H12N2

Conditions	Yield
With sodium hydroxide In ethanol at 130℃; for 144h; pH=7; High pressure;	60%

5,5'-dithiobis-(2-nitrobenzoic acid) (69-78-3) + (4E,8E,12E,16E)-4,8,13,17,21-pentamethyl-4,8,12,16,20-docosapentaen-1-thiol (118599-21-6) → 6-nitro-3-<(4E,8E,12E,16E)-4,8,13,17,21-pentamethyl-4,8,12,16,20-docosapentaenyldisulfamyl> benzoic acid

Conditions	Yield
In ethanol Ambient temperature;	57%

methanol (67-56-1) + 5,5'-dithiobis-(2-nitrobenzoic acid) (69-78-3) → methyl 2-(2-methoxycarbonyl-3-nitrophenyl)disulfanyl-6-nitrobenzoate (115215-73-1)

Conditions	Yield
With sulfuric acid Reflux;	57%
With thionyl chloride at 0℃; for 19h; Reflux;	55%
With dmap; dicyclohexyl-carbodiimide In dichloromethane at 20℃; for 20h; Inert atmosphere;

1,10-Phenanthroline (66-71-7) + 5,5'-dithiobis-(2-nitrobenzoic acid) (69-78-3) + manganese (II) acetate tetrahydrate (6156-78-1) → C14H6N2O8S2(2-)*Mn(2+)*C12H8N2

Conditions	Yield
With sodium hydroxide In ethanol; water at 130℃; for 144h; pH=7; High pressure;	56%

5,5'-dithiobis-(2-nitrobenzoic acid) (69-78-3) + water (7732-18-5) + manganese (II) acetate tetrahydrate (6156-78-1) + trans-1,2-bis(4-pyridyl)ethylene (1135-32-6) → C14H6N2O8S2(2-)*Mn(2+)*3H2O*C12H10N2

Conditions	Yield
With sodium hydroxide In ethanol at 130℃; for 144h; pH=7; High pressure;	55%

5,5'-dithiobis-(2-nitrobenzoic acid) (69-78-3) + thiocholesterol (1249-81-6) → 5-[(thiocholesteryl)thio]-2-nitrobenzoic acid (191990-34-8)

Conditions	Yield
With triethylamine In tetrahydrofuran at 20℃; for 14h;	51%

5,5'-dithiobis-(2-nitrobenzoic acid) (69-78-3) + 2-mercaptoethylamine hydrochloride (156-57-0) → S-<(3-carboxy-4-nitrophenyl)thio>-2-aminoethanethiol (71899-86-0)

Conditions	Yield
With sodium hydroxide In water at 20℃; for 2h; pH=7;	50%
With sodium hydroxide for 1h; Yield given;

5,5'-dithiobis-(2-nitrobenzoic acid) (69-78-3) + cucurbituril (80262-44-8) → C36H36N24O12*2C14H8N2O8S2*2H3N*2H2O

Conditions	Yield
With water; ammonium chloride at 120℃; for 6h; Time; Temperature; Autoclave;	50%

Octadecanethiol (2885-00-9) + 5,5'-dithiobis-(2-nitrobenzoic acid) (69-78-3) → 5-(Octadecyldithio)-2-nitrobenzoic acid (136860-97-4)

Conditions	Yield
With triethylamine In chloroform for 5h;	47%

5,5'-dithiobis-(2-nitrobenzoic acid) (69-78-3) + 4-t-butylbenzenethiol (2396-68-1) → 5-((4-(tert-butyl)phenyl)disulfaneyl)-2-nitrobenzoic acid

Conditions	Yield
In ethanol; water; acetonitrile at 20℃;	41%

3-mercaptopropionitrile (1001-58-7) + 5,5'-dithiobis-(2-nitrobenzoic acid) (69-78-3) → 5-(2-cyanoethyldithio)-2-nitrobenzoic acid (149997-68-2)

Conditions	Yield
With phosphate buffer for 0.5h; Ambient temperature;	40%

5,5'-dithiobis-(2-nitrobenzoic acid) (69-78-3) + Cysteamine (60-23-1) → S-<(3-carboxy-4-nitrophenyl)thio>-2-aminoethanethiol (71899-86-0)

Conditions	Yield
In water; acetonitrile for 14h; Ambient temperature;	40%
In water; acetonitrile	40%

5,5'-dithiobis-(2-nitrobenzoic acid) (69-78-3) + cucurbituril (80262-44-8) → C36H36N24O12*2C14H7N2O8S2(1-)*2H2O*Ba(2+)

Conditions	Yield
With BaCl2*10H2O In water at 120℃; for 6h; Autoclave;	40%

5,5'-dithiobis-(2-nitrobenzoic acid) (69-78-3) + 3-hydroxy-3,4-dihydrobenzotriazine-4-one (28230-32-2) → bis{3-O-[N-1,2,3-benzotriazin-4(3H)-one]-yl}-5,5'-dithiobis-2-nitrobenzoate (266685-49-8)

Conditions	Yield
With dicyclohexyl-carbodiimide In dichloromethane at 20℃; for 12h;	31%
With dicyclohexyl-carbodiimide In dichloromethane at 20℃; for 12h; Etherification;	31%

methanol (67-56-1) + cadmium(II) nitrate tetrhydrate + 5,5'-dithiobis-(2-nitrobenzoic acid) (69-78-3) → [Cd(5,5′-dithiobis(2-nitrobenzoate))(methanol)]n

Conditions	Yield
In 2-methyl-propan-1-ol at 85℃; Sealed tube; High pressure;	30%

Upstream product

• 77874-90-9 3-carboxylato-4-nitrothiophenoxide ion 
• 80-15-9 Cumene hydroperoxide 
• 15139-21-6 5-thio-2-nitrobenzoic acid 
• 6950-43-2 5-bromo-2-nitrobenzoic acid 
• 591-17-3 meta-bromotoluene 
• 75-91-2 tert.-butylhydroperoxide 
• 2516-95-2 5-chloro-2-nitrobenzoic acid 

Downstream Products

• 18430-02-9 3-carboxy-4-nitrobenzenethiolate 
• 629-45-8 dibutyl disulfide 
• 15139-21-6 5-thio-2-nitrobenzoic acid 
• 822-27-5 dioctyl disulfide 
• 5586-15-2 bis(2-naphthyl)disulfide 
• 77874-90-9 3-carboxylato-4-nitrothiophenoxide ion 
• 56-89-3 cysteine disulfide 
• 85908-82-3 C₃₅H₇₃NS₂*C₇H₅NO₄ 
• 39879-87-3 dicholest-5-en-3β-yl disulfide 
• 40055-98-9 oxidized glutathione 
• 18430-02-9 2-nitro-5-mercaptobenzoic acid anion 
• 247237-60-1 3-(3-carboxy-4-nitrophenylthio)-4-hydroxy-1-methyl-2(1H)-quinolone 

Relevant articles and documents

Investigation of solvent-dependent catalytic behaviour of hydrophobic guest artificial glutathione peroxidase using H2O2 and 3-carboxyl-4-nitrobenzenethiol as substrates

The investigation of the catalytic behaviour of a hydrophobic guest artificial glutathione peroxidase (GPx) (ADA-Te-OH) was carried out employing H2O2 and 3-carboxyl-4-nitrobenzenethiol (TNB) as substrates. The relation between the catalytic rate of ADA-Te-OH and the property of solvent used in the determination of catalytic activity was revealed. Typically, the co-solvents including ethanol, DMSO, DMF and CH3CN were employed in the determination of catalytic rates. It indicated that ADA-Te-OH exhibited the typical solventdependent catalytic behaviour. Especially, higher catalytic rate was observed when polar protic solvent (ethanol) was used compared with other co-solvents. It suggested that polar protic solvent was the appropriate co-solvent for the assay of catalytic activity of hydrophobic artificial glutathione peroxidase. Additionally, the strong polarity of polar aprotic solvent plays an important role in the enhancement of glutathione peroxidase catalytic activity. This study embodies well understanding of the catalytic behaviour of hydrophobic guest artificial glutathione peroxidase.

Selenosubtilisin as a glutathione peroxidase mimic

-

Construction of pH sensitive smart glutathione peroxidase (GPx) mimics based on pH responsive pseudorotaxanes

Two organoselenium compounds, both of which were modified with two primary amine groups, were designed and synthesized to mimic the catalytic properties of glutathione peroxidase (GPx). It was demonstrated that the catalytic mechanism of the diselenide organoselenium compound (compound 1) was a ping-pong mechanism while that of the selenide organoselenium compound (compound 2) was a sequential mechanism. The pH-controlled switching of the catalytic activities was achieved by controlling the formation and dissociation of the pseudorotaxanes based on the organoselenium compounds and cucurbit[6]uril (CB[6]). Moreover, the switching was reversible at pH between 7 and 9 for compound 1 or between 7 and 10 for compound 2.

Wavelength-Controlled Dynamic Metathesis: A Light-Driven Exchange Reaction between Disulfide and Diselenide Bonds

Wavelength-controlled dynamic processes are mostly based on light-triggered isomerization or the cleavage/formation of molecular connections. Control over dynamic metathesis reactions by different light wavelengths, which would be useful in controllable dynamic chemistry, has rarely been studied. Taking advantage of the different bond energies of disulfide and diselenide bonds, we have developed a wavelength-driven exchange reaction between disulfides and diselenides, which underwent metathesis under UV light to produce Se?S bonds. When irradiated with visible light, the Se?S bonds were reversed back to those of the original reactants. The conversion of the exchange depends on the wavelength of the incident light. This light-driven metathesis chemistry was also applied to tune the mechanical properties of polymer materials. The visible-light-induced reverse reaction was compatible with reductant-catalyzed disulfide/diselenide metathesis, and could be utilized to develop a dissipative system with light as the energy input.