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New Water-Soluble Phosphines as Reductants of Peptide and Protein Disulfide Bonds:  Reactivity and Membrane Permeability

TCEP 化学 二硫苏糖醇 反应性(心理学) 磷化氢 特里斯 磷化氢 生物结合 位阻效应 药物化学 有机化学 组合化学 生物化学 医学 替代医学 病理 催化作用 阻燃剂
作者
Daniel J. Cline,Sarah E. Redding,Stephen G. Brohawn,James N. Psathas,Joel P. Schneider,Colin Thorpe
出处
期刊:Biochemistry [American Chemical Society]
卷期号:43 (48): 15195-15203 被引量:198
标识
DOI:10.1021/bi048329a
摘要

Tris(2-carboxyethyl)phosphine (TCEP) is a widely used substitute for dithiothreitol (DTT) in the reduction of disulfide bonds in biochemical systems. Although TCEP has been recently shown to be a substrate of the flavin-dependent sulfhydryl oxidases, there is little quantitative information concerning the rate by which TCEP reduces other peptidic disulfide bonds. In this study, mono-, di-, and trimethyl ester analogues of TCEP were synthesized to evaluate the role of carboxylate anions in the reduction mechanism, and to expand the range of phosphine reductants. The effectiveness of all four phosphines relative to DTT has been determined using model disulfides, including a fluorescent disulfide-containing peptide (H(3)N(+)-VTWCGACKM-NH(2)), and with protein disulfide bonds in thioredoxin and sulfhydryl oxidase. Mono-, di-, and trimethyl esters exhibit phosphorus pK values of 6.8, 5.8, and 4.7, respectively, extending their reactivity with the model peptide to correspondingly lower pH values relative to that of TCEP (pK = 7.6). At pH 5.0, the order of reactivity is as follows: trimethyl- > dimethyl- > monomethyl- > TCEP >> DTT; tmTCEP is 35-fold more reactive than TCEP, and DTT is essentially unreactive. Esterification also increases lipophilicity, allowing tmTCEP to penetrate phospholipid bilayers rapidly (>30-fold faster than DTT), whereas the parent TCEP is impermeant. Although more reactive than DTT toward small-molecule disulfides at pH 7.5, all phosphines are markedly less reactive toward protein disulfides at this pH. Molecular modeling suggests that the nucleophilic phosphorus of TCEP is more sterically crowded than the thiolate of DTT, contributing to the lower reactivity of the phosphine with protein disulfides. In sum, these data suggest that there is considerable scope for the synthesis of phosphine analogues tailored for specific applications in biological systems.
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