Dithiothreitol (DTT) is an

organosulfur compound Organosulfur chemistry is the study of the properties and synthesis of organosulfur compounds, which are organic compounds that contain sulfur. They are often associated with foul odors, but many of the sweetest compounds known are organosulfur der ...

with the formula . A colorless compound, it is classified as a dithiol and a

diol A diol is a chemical compound containing two hydroxyl groups ( groups). An aliphatic diol may also be called a glycol. This pairing of functional groups is pervasive, and many subcategories have been identified. They are used as protecting gro ...

. DTT is

redox Redox ( , , reduction–oxidation or oxidation–reduction) is a type of chemical reaction in which the oxidation states of the reactants change. Oxidation is the loss of electrons or an increase in the oxidation state, while reduction is t ...

reagent also known as Cleland's reagent, after W. Wallace Cleland. The reagent is commonly used in its racemic form. Its name derives from the four-carbon

sugar Sugar is the generic name for sweet-tasting, soluble carbohydrates, many of which are used in food. Simple sugars, also called monosaccharides, include glucose Glucose is a sugar with the Chemical formula#Molecular formula, molecul ...

, threose. DTT has an epimeric ('sister') compound, dithioerythritol (DTE).

Synthesis

The traditional route to dithiothreitol (and its isomer dithioerythritol) is sulfidation of the extremely lachrymatory . Modern industrial syntheses instead use related epoxides and hydrogen sulfide.

Reducing agent

DTT is a

reducing agent In chemistry, a reducing agent (also known as a reductant, reducer, or electron donor) is a chemical species that "donates" an electron to an (called the , , , or ). Examples of substances that are common reducing agents include hydrogen, carbon ...

; once oxidized, it forms a stable six-membered ring with an internal

disulfide bond In chemistry, a disulfide (or disulphide in British English) is a compound containing a functional group or the anion. The linkage is also called an SS-bond or sometimes a disulfide bridge and usually derived from two thiol groups. In inor ...

. It has a redox potential of −0.33 V at pH 7. The reduction of a typical disulfide bond proceeds by two sequential

thiol-disulfide exchange In chemistry, a disulfide (or disulphide in British English) is a compound containing a functional group or the anion. The linkage is also called an SS-bond or sometimes a disulfide bridge and usually derived from two thiol groups. In inor ...

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

. 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 In organic chemistry, a thiol (; ), or thiol derivative, is any organosulfur compound of the form , where R represents an alkyl or other organic substituent. The functional group itself is referred to as either a thiol group or a sulfhydryl grou ...

form -SH is not); the pKa of the thiol groups is 9.2 and 10.1.

Applications

DTT is used as a reducing or "deprotecting" agent for thiolated DNA. The terminal sulfur atoms of thiolated

DNA Deoxyribonucleic acid (; DNA) is a polymer composed of two polynucleotide chains that coil around each other to form a double helix. The polymer carries genetic instructions for the development, functioning, growth and reproduction of al ...

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 In chemical analysis, chromatography is a laboratory technique for the Separation process, separation of a mixture into its components. The mixture is dissolved in a fluid solvent (gas or liquid) called the ''mobile phase'', which carries it ...

(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

s of

protein Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residue (biochemistry), residues. Proteins perform a vast array of functions within organisms, including Enzyme catalysis, catalysing metab ...

s and, more generally, to prevent ''intramolecular'' and ''intermolecular'' disulfide bonds from forming between

cysteine Cysteine (; symbol Cys or C) is a semiessential proteinogenic amino acid with the chemical formula, formula . The thiol side chain in cysteine enables the formation of Disulfide, disulfide bonds, and often participates in enzymatic reactions as ...

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

temperature Temperature is a physical quantity that quantitatively expresses the attribute of hotness or coldness. Temperature is measurement, measured with a thermometer. It reflects the average kinetic energy of the vibrating and colliding atoms making ...

s, or in the presence of a strong denaturant such as 6 M guanidinium chloride, 8 M

urea Urea, also called carbamide (because it is a diamide of carbonic acid), is an organic compound with chemical formula . This amide has two Amine, amino groups (–) joined by a carbonyl functional group (–C(=O)–). It is thus the simplest am ...

, or 1% sodium dodecylsulfate). DTT is oftentimes used along with sodium dodecylsulfate in SDS-PAGE to further denature proteins by reducing their disulfide bonds to allow for better separation of proteins during

electrophoresis Electrophoresis is the motion of charged dispersed particles or dissolved charged molecules relative to a fluid under the influence of a spatially uniform electric field. As a rule, these are zwitterions with a positive or negative net ch ...

. Because of the ability to reduce disulfide bonds, DTT can be used to denature CD38 on red blood cells. DTT will also denature antigens in the Kell, Lutheran, Dombrock, Cromer, Cartwright, LW and Knops blood group systems. 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 An oxidizing agent (also known as an oxidant, oxidizer, electron recipient, or electron acceptor) is a substance in a redox chemical reaction that gains or " accepts"/"receives" an electron from a (called the , , or ''electron donor''). In ot ...

. 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 In chemistry, an adduct (; alternatively, a contraction of "addition product") is a product of a direct addition of two or more distinct molecules, resulting in a single reaction product containing all atoms of all components. The resultant is ...

may be formed, i.e., the two sulfur atoms of DTT may form

s to different sulfur atoms; in such cases, DTT cannot cyclize since it has no such remaining free thiols.

Properties

DTT is oxidized by air. So it is normally stored and handled under inert atmosphere to minimize oxidation. The rate of air-oxidation is slower at low temperatures. Oxidized DTT exhibits a strong absorbance peak at 280 nm. Since thiols are less nucleophilic than their conjugate bases, thiolates, DTT becomes a less potent nucleophile as the pH falls. (2''S'')-2-Amino-1,4-dimercaptobutane ( dithiobutylamine or DTBA), a related dithiol reducing agent, somewhat overcomes this limitation of DTT. Tris(2-carboxyethyl)phosphine (

TCEP TCEP (tris(2-carboxyethyl)phosphine) is a reducing agent frequently used in biochemistry and molecular biology applications. It is often prepared and used as a hydrochloride salt (chemistry), salt (TCEP-HCl) with a molecular weight of 286.65 gra ...

) is an alternative reducing agent that is more stable and effective at low pH, but it is bulky and reduces cystines in folded proteins only slowly. DTT's half-life is 40 hours at pH 6.5 and 1.4 hours at pH 8.5 and 20 °C; its half-life decreases further as temperature increases. The presence of

EDTA Ethylenediaminetetraacetic acid (EDTA), also called EDTA acid, is an aminopolycarboxylic acid with the formula . This white, slightly water-soluble solid is widely used to bind to iron (Fe2+/Fe3+) and calcium ions (Ca2+), forming water-solubl ...

(ethylenediaminetetraacetic acid) to chelate divalent metal ions considerably extends the half-life of DTT in solution.

References

External links