Dithiothreitol (DTT) is the racemic (2S,3S;2R,3R) threo isomer of the compound 2,3-dihydroxy-1,4-dithiolbutane. DTT, also known as Cleland’s Reagent, is a cell-permeable, strong but mild reducing agent, with a low redox potential of -0.33 V at pH 7 (optimal pH range 7.1 to 8.0). This enables it to reduce practically all accessible biological disulfides, as well as to protect free thiols from oxidation. 

The reduction of disulfide-containing compounds by DTT is a two-step reaction. First, DTT forms a mixed disulfide with the target molecule in a reversible reaction, reducing one sulfur to the corresponding sulfhydryl. In the second step, intramolecular cyclization of DTT leads to an energetically favored six-membered cyclic disulfide. This drives the reaction to completion, reducing the second sulfur of the original disulfide bond. Consequently, DTT is required in much lower concentrations compared to monothiol reducing agents (e.g. glutathione or 2-mercaptoethanol). The latter ones are usually required in extreme excess to completely reduce target compounds.

Currently, DTT is being employed in the large-scale production of antibody tests against SARS-CoV-2.

Most commonly, DTT is used to reduce disulfide bonds in peptides and proteins. However, as with all reductants, DTT can only reduce disulfides if they are accessible. At low concentrations, DTT can be used to stabilize proteins with free thiol groups. Conversely, high concentrations of DTT are utilized in order to denature proteins, e.g. before performing an SDS-PAGE.

In recombinant protein expression, the formation of insoluble inclusion bodies is a frequently encountered problem. Dithiothreitol is an integral reagent, together with denaturants such as GdmCl, for the solubilization of proteins from inclusion bodies. 

DTT is also employed for the cleavage of disulfide-containing crosslinkers or modifications. For example, two macromolecules connected by a disulfide-bearing crosslinker may be separated again by mild reduction, if desired. This technique is frequently employed in the analysis of receptor-ligand interactions, as well as in the study of protein-protein interactions in vivo. 

Due to its strong reduction potential, DTT can also be used to reduce organic azides to their corresponding amines in a pH range of 7.2 to 9.0. Common conditions for azide reduction include Staudinger conditions using phosphines, catalytic hydrogenation, and reduction by stannous chloride. In contrast to reduction by DTT, however, none of those methods is both mild and selective, while allowing for the complete removal of reactants after completion. One current example is the reductive activation of Belyntic’s innovative PEC-Linker (see our recent News Article on the Peptide Easy Clean technology).

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References:

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• Enzyme activity and sulfhydryl status in rat renal cortex following mercuric chloride and dithiothreitol administration; D. R. Klonne and D. R. Johnson; Toxicol Lett 1988; 42: 199-205. https://doi.org/10.1016/0378-4274(88)90078-1
• Molecular mechanism for prevention of N-acetyl-p-benzoquinoneimine cytotoxicity by the permeable thiol drugs diethyldithiocarbamate and dithiothreitol; V. V. Lauriault and P. J. O'Brien; Mol Pharmacol 1991; 40: 125-34.
• Large scale, in situ isolation of periplasmic IGF-I from E. coli; R. A. Hart, P. M. Lester, D. H. Reifsnyder, J. R. Ogez and S. E. Builder; Biotechnology (N Y) 1994; 12: 1113-7. https://doi.org/10.1038/nbt1194-1113 
• Kinetics and Equilibria of the Thiol/Disulfide Exchange Reactions of Somatostatin with Glutathione; D. L. Rabenstein and K. H. Weaver; J Org Chem 1996; 61: 7391-7397. https://doi.org/10.1021/jo960917+
• Folding proteins; R. Rudolph, G. Böhm, H. Lilie and R. Jaenicke; Protein function: A practical approach 1997; 2: 57-99. 
• Refolding of recombinant proteins; E. De Bemardez Clark; Curr. Opin. Biotechnol 1998; 9: 157-163. 
• Advances in refolding of proteins produced in E. coli; H. Lilie, E. Schwarz and R. Rudolph; Curr Opin Biotechnol 1998; 9: 497-501. https://doi.org/10.1016/s0958-1669(98)80035-9 
• Functional Targets for Bioconjugation; G. T. Hermanson; Bioconjugate Techniques G. T. Hermanson 2013: 127-228. https://doi.org/10.1016/b978-0-12-382239-0.00002-9 
• Kinetics and mechanisms of thiol-disulfide exchange covering direct substitution and thiol oxidation-mediated pathways; P. Nagy; Antioxid Redox Signal 2013; 18: 1623-41. https://doi.org/10.1089/ars.2012.4973