Background: The unique hypervariable C-terminal region (HVR) of K-Ras4B, one of the most frequently mutated proteins in many powerful cancers, contains a C-terminal farnesylated and methylated Cys and a poly-lysine motif, which decides the association of K-Ras4B to the inner leaflet of plasma membrane for activating the downstream signaling activity. In our previous work, we inserted an additional Cys in K-Ras4B HVR peptide synthesis for NCL in the semi-synthesis of K-Ras4b protein, but it is not suitable for application in protein dimerization research. The recently developed selenocysteine (Sec, U) mediated native chemical ligation reaction followed by selective deselenization, which can help to broaden the scope of protein synthesis, requires the generation of the peptide fragment with an N-terminal Sec.

Objective: To synthesize K-Ras4B HVR peptide containing both N-terminal Sec and C-terminal farnesylated and methylated Cys to achieve traceless protein semi-synthesis.

Methods and Results: We have developed a facile synthesis approach for producing Boc-Sec)2-OH using economic Se powder, which can facilitate scaling up preparation of peptides containing Sec at the N-terminus. Furthermore, we synthesized K-Ras4B HVR peptide containing selenocystine by utilization of Boc-Sec)2-OH. Finally, we took K-Ras4B HVR peptide as an example to test the compatibility of farnesylation reaction at Cys with the N-terminal Sec)2, and the farnesyl group was successfully added to the thiol group of Cys.

背景：K-Ras4B 的独特高变 C 端区域 (HVR) 是许多强大癌症中最常见的突变蛋白之一，包含 C 端法呢基化和甲基化的 Cys 和多聚赖氨酸基序，它决定了K-Ras4B 到质膜的内小叶以激活下游信号活动。在我们之前的工作中，我们在 K-Ras4b 蛋白的半合成中为 NCL 的 K-Ras4B HVR 肽合成插入了一个额外的 Cys，但它不适合应用于蛋白质二聚化研究。最近开发的硒代半胱氨酸 (Sec, U) 介导的天然化学连接反应，然后是选择性脱硒，这有助于扩大蛋白质合成的范围，需要生成具有 N 端 Sec 的肽片段。

目的：合成含有N端Sec和C端法呢基化和甲基化Cys的K-Ras4B HVR肽，实现无痕蛋白半合成。

方法和结果：我们开发了一种使用经济的硒粉生产 Boc-Sec)2-OH 的简便合成方法，这有助于扩大在 N 端含有 Sec 的肽的制备。此外，我们通过利用 Boc-Sec)2-OH 合成了含有硒代胱氨酸的 K-Ras4B HVR 肽。最后，我们以K-Ras4B HVR肽为例，测试了Cys处的法呢基化反应与N端Sec)2的相容性，并成功将法尼基加入到Cys的巯基中。