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. Wallin , Ronald T. Pyrazine-derived disulfide-reducing agent for chemical biology. Chemical Communications , 50 67 , Angewandte Chemie , 49 , Angewandte Chemie International Edition , 52 49 , A highly selective ratiometric fluorescent probe for 1,4-dithiothreitol DTT detection.
Beck , Shen-Chieh Chou. The Structural Diversity of Phthalides from the Apiaceae. Journal of Natural Products , 70 5 , Using mass spectrometry to study the photo-affinity labeling of protein tyrosine phosphatase 1B.
International Journal of Mass Spectrometry , 2 , Study of the disulfide reduction of denatured proteins by liquid chromatography coupled with on-line cold-vapor-generation atomic-fluorescence spectrometry LC—CVGAFS.
Analytical and Bioanalytical Chemistry , 2 , Journal of Biological Chemistry , 46 , M W. Wallace Cleland. Skorey , Brian P. Kennedy , Richard W. Friesen , Chidambaram Ramachandran. Read more about how to correctly acknowledge RSC content. Fetching data from CrossRef. This may take some time to load. Loading related content.
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