DL-Dithiothreitol CAS 3483-12-3

DL-Dithiothreitol (DTT for short) is a small molecule organic reducing agent with the chemical formula C4H10O2S2. It is a linear molecule in the reduced state, and becomes a six membered ring structure containing disulfide bonds after oxidation. The name dithiothreitol derives from threose, a four carbon monosaccharide. The isomer of DTT is dithioerythritol (DTE), which is the C3 epimer of DTT. Soluble in water.

Because it is easy to be oxidized by air, the stability of DTT is poor; However, freezing storage or treatment in inert gas can prolong its service life. Due to the low nucleophilicity of protonated sulfur, the effective reduction of DTT decreases with the decrease of pH; Tris (2-carboxyethyl) phosphate HCl (TCEP hydrochloride) can be used as a substitute for DTT at low pH and is more stable than DTT.

1. One of the uses of DTT is as a reductant and deprotector of thiolated DNA. Sulfur atoms at the end of thiolated DNA tend to form dimers in solution, especially in the presence of oxygen. This dimerization greatly reduces the efficiency of some coupling reaction experiments (such as DNA fixation in biosensors); The addition of DTT in the DNA solution can reduce the DNA dimerization by removing it after a period of reaction.

2. DTT is also often used for the reduction of disulfide bonds in proteins, which can be used to prevent the formation of protein intramolecular or intermolecular disulfide bonds between cysteine in proteins. However, DTT is often unable to reduce the disulfide bonds embedded in the protein structure (inaccessible to solvents). The reduction of such disulfide bonds often requires protein denaturation (high temperature heating or adding denaturants, such as 6M guanidine hydrochloride, 8M urea or 1% SDS). On the contrary, according to the different reduction rates of disulfide bonds in the presence of DTT, the depth of its embedment can be determined.

For the new synthesis method of dithiothreitol, the raw material consumption is calculated according to the theoretical product yield of 277.6g, including the following steps:

a. Add 162.5g (1.80mol) of 1,4-butenediol into the reaction kettle, slowly drip 316.8g (1.98mol) of liquid bromine under the stirring condition, stir for 16 hours under the temperature of 25 ℃ after dropping, stand still for layering, take the supernatant, and then obtain 2,3-dibromo-1,4-butanediol. Store it in a tank with the temperature lower than 30 ℃ for standby;

b. In the supernatant obtained in step a, 651.2g (4.88mol) of sodium hydroxide solution with a mass percent concentration of 30% is added, and the reaction is stirred at a temperature lower than 20 ℃ for 16 hours to obtain ethylene oxide;

c. Add 155.67g (2.045mol) of thioacetic acid into the reaction solution obtained in step b, stir and react for 20 hours, distill under reduced pressure, and crystallize to obtain dithiothreitol diacetate;

d. In the crystallization obtained in step c, 190.9g (1.432mol) of sodium hydroxide solution with a mass percent concentration of 30% is added, stirred for reaction for 4 hours, cooled to room temperature, adjusted pH to 7.5~8.0 with hydrochloric acid solution with a mass percent concentration of 3%, extracted with 300mL of ethyl acetate, combined the organic layers, distilled under normal pressure to remove ethyl acetate, and then distilled the residual concentrated solution under reduced pressure at a vacuum of 15mmHg and a temperature of 180~200 ℃, The distillate with temperature between 130~140 ℃ was collected and cooled to room temperature to obtain 100g of white solid dithiothreitol with yield of 36.02%.

DTT is a strong reducing agent, and its reducibility is largely due to the conformational stability of its oxidized six membered ring (containing disulfide bond). Its redox potential is -0.33 V at pH 7. The reduction of a typical disulfide bond by dithiothreitol consists of two consecutive mercapto disulfide bond exchange reactions.

Among them, the intermediate state formed in the first step of the reaction is very unstable, because the second sulfhydryl group on the DTT tends to connect with the oxidized sulfur atom, so that the intermediate state is quickly transformed into the cyclic oxidation structure of the DTT, thus completing the reduction of the disulfide bond.

The reducing power of DTT is affected by the pH value, and it can only play a reducing role when the pH value is greater than 7. This is because only the protonated mercaptan anion (- S –) has reactivity, while mercaptan (- SH) does not; The pKa of thiol group is generally~8.3.

Because it is easy to be oxidized by air, the stability of DTT is poor; However, freezing storage or treatment in inert gas can prolong its service life. Due to the low nucleophilicity of protonated sulfur, the effective reduction of DTT decreases with the decrease of pH; Tris (2-carboxyethyl) phosphate HCl (TCEP hydrochloride) can be used as a substitute for DTT at low pH and is more stable than DTT.

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