Inprovement of Thioester Method and Synthesis of Cysteine-Containing Proteins

为了扩展硫酯法，我们探索了制备S-保护肽硫酯的途径。通过固相法的Boc策略获得的保护肽树脂，通过低HF或高HF处理制备S-保护肽硫酯。今年，开发了一种更有效的制备S-保护肽硫酯的方法。在该方法中，通过使用Npys-氨基酸延长肽链。半胱氨酸残基由Npys-Cys（Tmb）（Tmb：2，4，6-三甲基苄基）引入。用试剂K处理保护的肽树脂，得到S-保护的肽硫酯。基于此方法，合成了含半胱氨酸的肽硫酯，并将其用于酿酒酵母（Saccharomycescerevisiae）DNA指导的RNA聚合酶Ⅱ（RPSc（299-410））中112个氨基酸残基的类巴马酶结构域的合成。成功合成了RPSc（299-410）。该策略也应用于HIV-1的达特蛋白的制备，该蛋白具有7个半胱氨酸残基，也是通过硫酯法合成的。达特蛋白质与锌离子的相互作用正在研究中，硫酯法同样适用于含半胱氨酸蛋白质和不含半胱氨酸蛋白质的分离。

In order to expand the thioester method, we searched the route for the preparation of S-proted two methods for the preparation of S-protected peptide thioesters via low- or high-HF treatment of protected peptide resins obtained by Boc strategy of a solid-phase method. This year, a more efficient method for the preparation of S-protected peptide thioester wwas developed. In this method, peptide chain was elongated by using Npys-amino acids. A cysteine residue is introduced by Npys-Cys(Tmb) (Tmb : 2,4,6-trimethylbenzyl). Aprotected peptide resin was treated by reagent K to give an S-protected peptide thioester. The Tmb group on the mercapt group was stable during reagent K treatment and segment condensation.Vased on this preparation strategy, cysteine-containing peptide thioesters were synthesized and used for the syntheses of the barmase-like domain ( 112 amino acid residues) in DNA-directed RNA polymerase II of Saccharomyces cerevisiae (RPSc(299-410)). RPSc(299-410) was successfully synthesided. This strateby was also applied to the preparation of tAT protein of HIV-1, which has 7 cysteine residues, was also synthesized by the thioester method. the interaction between TAT protein and zinc ions is now under investigation.To conclude, the thioester method can be applied to the synthsis of proteins with cysteine residues as sell as without them.