Small molecule-based electrophilic compounds such as 1-chloro-2,4-dinitrobenzene (CDNB)a nd 1-chloro-4nitrobenzene (CNB) are currently being used as inhibitors of cysteine-and selenocysteine-containing proteins. CDNB has been used extensively to determine the activity of glutathione S-transferase and to depleteg lutathione (GSH) in mammalianc ells. Also, CDNB has been shown to irreversibly inhibit thioredoxin reductase (TrxR), as elenoenzyme that catalyses the reduction of thioredoxin (Trx). Mammalian TrxR has aC -terminal active site motif, Gly-Cys-Sec-Gly,a nd both the cysteine and selenocysteine residues could be the targets of the electrophilicr eagents. In this paper we report on the stability of aseries of cysteine and selenocysteine deriva-tives that can be considered as modelsf or the selenoenzyme-inhibitor complexes.W es how that these derivatives react with H 2 O 2 to generate the corresponding selenoxides, which undergo spontaneouse limination to produce dehydroalanine. In contrast, the cysteine derivatives are stable towards suche limination reactions. We also demonstrate, for the first time, that the arylselenium speciese liminated from the selenocysteine derivatives exhibit significant redox activity by catalysing the reduction of H 2 O 2 in the presence of GSH (GPx (glutathione peroxidase)-like activity), which suggests that such redoxm odulatory activity of selenium compounds may have as ignificant effect on the cellular redox state during the inhibition of selenoproteins. Scheme1.(A) Chemical structures of somec ommonselenoenzyme inhibitors. (B) Mechanism of the inhibition of mammalian thioredoxin reductase (TrxR) by CDNB. The covalent bondsf ormed between TrxR and inhibitor molecules deactivate the enzyme.[a] K.Scheme2.Starting materials 1-3 used for the synthesis of the cysteine and selenocysteine derivatives and the reaction of compound 1 with thiols 4-6 to produce the aryl-substituted cysteine derivatives 7-9,r espectively. Reagentsa nd conditions: a) K 2 CO 3 ,DMF,08C, 5min, 27 8C, 6h;b)10% NaH 2 PO 4 ,EtOAc, 50 8C, 2h.
The S-acetamidomethyl (Acm) or trityl (Trt) protecting groups are widely used in the chemical synthesis of peptides that contain one or more disulfide bonds. Treatment of peptides containing S-Acm protecting group with iodine results in simultaneous removal of the sulfhydryl protecting group and disulfide formation. However, the excess iodine needs to be quenched or adsorbed as quickly as possible after completion of the disulfide bond formation in order to minimize side reactions that are often associated with the iodination step. We report here a simple method for simultaneous quenching and removal of iodine and isolation of disulphide bridge peptides. The use of excess inexpensive anion exchange resin to the oxidized peptide from the aqueous acetic acid/methanol solution affords quantitative removal of iodine and other color impurities. This improves the resin life time of expensive chromatography media that is used in preparative HPLC column during the purification of peptide using preparative HPLC. Further, it is very useful for the conversion of TFA salt to acetate in situ. It was successfully applied commercially, to the large scale synthesis of various peptides including Desmopressin, Oxytocin, and Octreotide. This new approach offers significant advantages such as more simple utility, minimal side reactions, large scale synthesis of peptide drugs, and greater cost effectiveness.
Selenocysteine (Sec), the 21 st proteinogenic amino acid, is inserted co-translationally into number of natural proteins. It is coded by a dual function stop codon UGA (opal). It is a redox active amino acid found at the active sites of several enzymes that are involved in oxidation-reduction reactions. These enzymes include the three major mammalian selenoproteins glutathione peroxidase (GPx), thioredoxin reductase (TrxR), and iodothyronine deiodinase (Dio). Although Sec is structurally similar to its sulfur analogue cysteine (Cys), the lower pK a of the selenol group in Sec as compared to that of Cys and the interesting redox properties of the selenium atom in peptides and proteins play crucial roles in redox catalysis. However, the chemical synthesis of Sec-containing peptides has been a difficult task. In this paper, we report on a new method for the synthesis of Sec and Sec-containing peptides using dehydroalanine (Dha) as a building block.
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