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Dithiothreitol DTT is the standard reagent for reducing disulfide bonds between and within biological molecules. Herein, we report on 2 S amino-1,4-dimercaptobutane dithiobutylamine or DTBA , a dithiol that can be synthesized from l -aspartic acid in a few high-yielding steps that are amenable to a large-scale process. The amino group of DTBA enables its isolation by cation-exchange and facilitates its conjugation.
These attributes indicate that DTBA is a superior reagent for reducing disulfide bonds in aqueous solution. Value is from ref Values are from ref 3f. Values are from ref 3a. At physiological pH, DTT is a sluggish reducing agent.
The reactivity of a dithiol is governed by the lower of its two thiol p K a values. Moreover, we sought a reagent that could be accessed in high yield from an inexpensive source. We envisioned that 2 S amino-1,4-dimercaptobutane dithiobutylamine or DTBA; Table 1 could fulfill our physicochemical criteria, and be synthesized from l -aspartic acid, which is an abundant amino acid. A five-step route commenced with the esterification of the amino acid and protection of its amino group.
Reduction with lithium aluminum hydride yielded a diol, which was subjected to Mitsunobu conditions to install the requisite sulfur functionality. In both routes, the product of every step is a white solid. DTBA has desirable physicochemical attributes. Its HCl salt is a nearly odorless white solid with high solubility in water. DTBA is an efficacious reducing agent for disulfide bonds in small molecules.
At pH 7. Figure 1. DTBA is also an efficacious reducing agent for disulfide bonds in proteins. A cysteine residue resides within the active site of papain Cys25 and near that of creatine kinase Cys Forming a mixed disulfide with those cysteine residues is known to eliminate their enzymatic activities.
The active site of papain is hydrophobic like its substrates, though there is an anionic region nearby Figure 2 A. In contrast, the two reagents reduce a disulfide bond near the cationic active site of creatine kinase at a similar rate. Figure 2. The amino group of DTBA confers additional benefits. For example, a disulfide-reducing agent that can be readily isolated, regenerated, and reused incurs less cost and generates less waste.
We also note that the amino group of DTBA enables its covalent attachment to a soluble molecule, resin, or surface by simple reactions, such as reductive amination which preserves the cationic charge or N-acylation. We conclude that the attributes of DTBA could enable it to supplant DTT as the preferred reagent for reducing disulfide bonds in biomolecules. Experimental protocols and analytical data. Such files may be downloaded by article for research use if there is a public use license linked to the relevant article, that license may permit other uses.
We are grateful to Professor W. Cleland and S. Johnston for enabling advice, and to N. McElfresh for preliminary work on this project. Louis, MO. Tris 2-carboxyethyl phosphine TCEP is more potent than DTT at reducing disulfide bonds between small molecules 2e but not within proteins. Another commercial dithiol, bis 2-mercaptoethyl sulfone BMS , has low thiol p K a values of 7. The amino group of DTBA is assumed to be cationic throughout its pH-titration, as cysteamine has an amino p K a of View Author Information.
Cite this: J. ACS AuthorChoice. Article Views Altmetric -. Citations Abstract High Resolution Image. Physical Properties of Disulfide-Reducing Agents. Table a Value is from ref Table b Values are from ref 3f. Table c Value is from ref Table d Values are from ref 3a.
Table e Value is from ref High Resolution Image. Supporting Information. Author Information. Ronald T. John C. Michael J. The authors declare no competing financial interest. Proteomics , 4 , — Google Scholar There is no corresponding record for this reference. Because of its low oxidn. Since this compd. Google Scholar There is no corresponding record for this reference.
Methods Enzymol. The rate consts. The max. At M concn. A convenient synthesis of a mixt. Free energies, transition structures, charge densities, and solvent effects along the reaction pathway have been detd. Mechanistic results agree with exptl. The transition structures have the charge d. The charge densities allow the authors to rationalize the solvent effects. As transition structures have the charge d. The effect can be so dramatic that disulfide exchange inside the active site of ribonucleotide reductase is estd.
It was also found that attack by thiol is much faster than previously assumed, if mediated through water chains. Although the present results, as well as exptl. There is no corresponding record for this reference. Proteins , 4 , 99 — Google Scholar There is no corresponding record for this reference. Tetrahedron Lett.
FEBS Lett. The reduction of a disulfide bond is followed by two sequential thiol-disulfide exchange reactions see Fig. The reduction generally continues past the mixed-disulfide species due to the second thiol in DTT having an increased tendency to close the ring.
This causes the formation of an oxidized DTT and leaves behind a reduced disulfide bond. The thiol groups have pKa values of 9. DTT is highly soluble in water, forming a clear solution. DTT is also soluble in the following:. Dithiothreitol can substitute for 2-mercaptoethanol in most applications. DTT solutions should be prepared fresh daily. If improperly stored including room temperature and solution forms its reducing ability may be reduced.
Exposure to air should be minimized, even though DTT has a lower tendency to be oxidized directly by air than other reducing agents. Note: DTT becomes less a less potent reducing agent with decreasing pH levels. DTT is commonly used in the study of disulfide exchange reactions to reduce the disulfide bonds of proteins and reconstruct the proteins before electrophoresis analysis.
The process removing DTT is performed via desalting procedures such as dialysis or gel-filtration. DTT stops the formation of both intra- and inter-molecular disulfide bonds between cysteine residues. Disulfide bonds that are inaccessible to the solvent can be further targeted using additional denaturing techniques such as increased temperature or the addition of a powerful denaturant. The optimal pH range for DTT is between 7.
DTT is well stable longer shelf life as a powder than 2-mercaptoethanol , however stock solutions must be used immediately and any remaining solution discarded. Mini Cart No products in the cart.
Figure 1. Reaction DTT participates to disulfide exchange reaction that drives its major applications. Applications As a reducing agent , it is used to reduce the disulfide bonds of proteins and peptides. It can be antioxidant agent , be used to produce biofuel , and lead apoptosis As a oxidizing agent , its principal advantage is that effectively no mixed-disulfide species are populated, in contrast to other agents such as glutathione.
Tips for Research DTT replaces in most applications the very pungent 2-mercaptoethanol.
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