Dithiothreitol

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Dithiothreitol[1]
Dithiothreitol.png
Identifiers
CAS number 3483-12-3 YesY
PubChem 446094
ChemSpider 393541 YesY
UNII T8ID5YZU6Y YesY
DrugBank DB04447
ChEBI CHEBI:42170 YesY
ChEMBL CHEMBL406270 N
Jmol-3D images Image 1
Properties
Molecular formula C4H10O2S2
Molar mass 154.25 g mol−1
Appearance White solid
Melting point 42–43 °C
Boiling point 125–130 °C at 2 mmHg
Solubility in water Soluble
Except where noted otherwise, data are given for materials in their standard state (at 25 °C (77 °F), 100 kPa)
 N (verify) (what is: YesY/N?)
Infobox references

Dithiothreitol (DTT) is the common name for a small-molecule redox reagent known as Cleland's reagent.[2] DTT's formula is C4H10O2S2 and the molecular structure of its reduced form is shown at the right; its oxidized form is a disulfide bonded 6-membered ring (shown below). Its name derives from the four-carbon sugar, threose. DTT has an epimeric ('sister') compound, dithioerythritol (DTE).

Reducing agent[edit]

DTT is an unusually strong reducing agent, because once oxidized, it forms a stable six-membered ring with an internal disulfide bond. It has a redox potential of -0.33 V at pH 7.[1] The reduction of a typical disulfide bond proceeds by two sequential thiol-disulfide exchange reactions and is illustrated below. The reduction usually does not stop at the mixed-disulfide species because the second thiol of DTT has a high propensity to close the ring, forming oxidized DTT and leaving behind a reduced disulfide bond. The reducing power of DTT is limited to pH values above 7, since only the negatively charged thiolate form -S is reactive (the protonated thiol form -SH is not); the pKa of the thiol groups is 9.2 and 10.1.

Reduction of a typical disulfide bond by DTT via two sequential thiol-disulfide exchange reactions.


Applications[edit]

A common use of DTT is as a reducing or "deprotecting" agent for thiolated DNA. The terminal sulfur atoms of thiolated DNA have a tendency to form dimers in solution, especially in the presence of oxygen. Dimerization greatly lowers the efficiency of subsequent coupling reactions such as DNA immobilization on gold in biosensors. Typically DTT is mixed with a DNA solution and allowed to react, and then is removed by filtration (for the solid catalyst) or by chromatography (for the liquid form). The DTT removal procedure is often called "desalting." Generally, DTT is used as a protecting agent that prevents oxidation of thiol groups.

DTT is frequently used to reduce the disulfide bonds of proteins and, more generally, to prevent intramolecular and intermolecular disulfide bonds from forming between cysteine residues of proteins. However, even DTT cannot reduce buried (solvent-inaccessible) disulfide bonds, so reduction of disulfide bonds is sometimes carried out under denaturing conditions (e.g., at high temperatures, or in the presence of a strong denaturant such as 6 M guanidinium hydrochloride, 8 M urea, or 1% sodium dodecylsulfate). Conversely, the solvent exposure of different disulfide bonds can be assayed by their rate of reduction in the presence of DTT.

DTT can also be used as an oxidizing agent. Its principal advantage is that effectively no mixed-disulfide species are populated, in contrast to other agents such as glutathione. In very rare cases, a DTT adduct may be formed, i.e., the two sulfur atoms of DTT may form disulfide bonds to different sulfur atoms; in such cases, DTT cannot cyclize since it has no remaining free thiols.

Properties[edit]

Due to air oxidation, DTT is a relatively unstable compound whose useful life can be extended by refrigeration and handling in an inert atmosphere. Oxidation presents further complications as oxidized DTT exhibits a strong absorbance peak at 260 nm. Since protonated sulfurs have lowered nucleophilicities, DTT becomes less potent as the pH lowers. (2S)-2-amino-1,4-dimercaptobutane (dithiobutylamine or DTBA) is a new dithiol reducing agent that somewhat overcomes this limitation of DTT.[3] Tris(2-carboxyethyl)phosphine HCl (TCEP hydrochloride) is an alternative reducing agent that is more stable and works even at low pH.

References[edit]

  1. ^ a b M.J.O'Neil, ed. by (2001). Merck Index : an encyclopedia of chemicals, drugs, & biologicals : 13th ed. (13. ed. ed.). United States: MERCK & CO INC. ISBN 0-911910-13-1. 
  2. ^ Cleland, W. W. (1964). "Dithiothreitol, a new protective reagent for SH groups". Biochemistry 3: 480–482. doi:10.1021/bi00892a002. PMID 14192894. 
  3. ^ Lukesh, III, J. C.; Palte, M. J.; Raines, R. T. (2012). "A potent, versatile reducing agent from aspartic acid". J. Am. Chem. Soc. 134 (9): 4057–4059. doi:10.1021/ja211931f. PMC 3353773. PMID 22353145.