15-6375 & 15-7400: Reducing Agents for Molecular Diagnostics
Two new odourless, water soluble compounds useful as reducing agents for neutral sulfhydryl compounds are now available from Strem Chemicals
Tris(3-hydroxypropyl)phosphine (THP) and Tris(2-carboxyethyl)phosphine, hydrochloride (TCEP) are odorless, water soluble, transparent compounds that are used as neutral sulfhydryl reducing agents. They are more resistant to air oxidation than dithiothreitol (DTT) and have a greater reducing capacity than DTT. In addition, THP and TCEP are suitable for use in immobilized metal affinity chromatography because they do not reduce the metals involved.
To maintain biological activity in biological systems, it is important that the thiol groups in peptides and proteins remain in a reduced state. Thiols, such as 2-mercaptoethanol and DTT, are commonly used as disulfide reductants. However, DTT can interfere in the desired reactions of SH groups and it can also be oxidized by metal-affinity columns. Therefore it must be removed prior to peptide modification or purification.
On the other hand, trialkylphosphines exhibit high selectivity for disulfide bonds, but are generally not water soluble, and have an unpleasant odor. However, the high water solubility, and neutral odor, of THP and TCEP make these two phosphines ideal alternatives to DTT. Unlike DTT, they generally do not need to be removed prior to thiol modification. THP has the added advantage of being effective over a wide pH range.1
THP and TCEP are used to break disulfide bonds within and between proteins in a wide range of biological applications. These applications include protein cleavage and/or precipitation for molecular diagnostics, gene chips and microarrays. In fact, THP and TCEP are both found in numerous enzyme kits for biological processing, such as protein extraction2, specific amino acid sequence cleavage3, and protein in-gel tryptic digestion.4
THP is also useful for the removal of protecting groups from S-protected cysteine5, the deoxygenation of sulfoxides, N-oxides, and other sulfur and nitrogen compounds6. It is also a reagent for peptide synthesis, particularly in Mitsunobu and Staudinger reactions7. TCEP, meanwhile, does not impede maleimide attachment, and is suitable as a reagent for the quantitative analysis of sulfides, disulfides, hypochlorite ions, iodine, iodate ions, sulfoxides, N-oxides, azides8, and the selective reduction of disulfides for organic synthesis.9
References:
1. US Patent Application 60/469,821.
2. Novagen Protocol TB245 Rev. E 0304, BugBusterstrem_com Protein Extraction Reagent; Novagen User Protocol TB316 Rev. B 0804, YeastBusterTM Protein Extraction Reagent.
3. Takara HRV 3C Protease Product Manual v201304Da.
4. Agilent 5188-2749Protein In-Gel Tryptic Digestion Kit Instructions.
5. Tris(hydroxypropyl)phosphine (THPP), New Applications in Organic Chemistry, Accessed 1/2/2014.
6. G.A. Olah, B.G.B. Gupta, S.. Narang, Synthesis 1978, 137.
7. (a). I. Bosch, F. Urpf, J. Vilarrasa, Chem. Commun., 91, 1995; (b) H. Fuwa e.a., Tetrahedron Lett., 2004, 45, 2323; J. S. Davies, J. Peptide Sci., 2003, 9, 471.
8. A.-M. Faucher, C. Grand-Maitre, Synth. Commun., 2003, 33, 3503.
9. J.A. Burns, J.C. Butler. J. Moran, G.M. Whitesides, J. Org. Chem., 1991, 56, 2648.
Products mentioned in this blog:
15-6375: Tris(3-hydroxypropyl)phosphine, min. 80%, CAS # 4706-17-6
15-7400: Tris(2-carboxyethyl)phosphine, hydrochloride, 99% TCEP, CAS # 51805-45-9
Keywords: Deprotection, peptide, protein, phosphine, Mitsunobu, Staudinger, sulfhydryl reducing agents, metal affinity chromatography
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