Site-specific biotinylation. A novel approach and its application to endothelin-1 analogs and PTH-analog.

S Natarajan, S M Festin, A Hedberg, E C Liu, D M Floyd, J T Hunt
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Abstract

We have developed an expeditious method for the incorporation of the biotinylaminocaproyl moiety on the epsilon-amino group of a lysine residue within a peptide chain in a site-specific manner. Using t-Boc chemistry for the solid phase synthesis approach and a base labile, acid stable protecting group (Fmoc-) for the epsilon-amino group of the target lysine, we prepared fully protected resin bound peptides which are site-specifically biotinylated. Following HF cleavage, the uniquely biotinylated peptides were obtained in a high degree of purity. Using this approach, a number of biotinylaminocaproyllysyl derivatives of a monocyclic Endothelin-1 analog were prepared. Synthesis of selected bicyclic analogs of high affinity monocycles led to the preparation of the bicyclic [Nle7]ET-1 analog containing epsilon-biotinylaminocaproyllysine at position-9. This peptide, with Kd = 0.08 nM, has 1000-fold higher affinity for the ETA receptor than the commercially available N alpha-biotinylated Endothelin-1. The general utility of this biotinylation methodology was demonstrated by the synthesis of a site-specifically biotinylated PTH analog which contained several side chain functionalized amino acid residues in its sequence. The synthetic method reported here is convergent in that it allows the facile variation of the length of the spacer and also offers the potential to introduce in a site specific manner other groups such as affinity labels and fluorescent tags.

因地制宜生物素酰化。内皮素-1类似物和甲状旁腺素类似物的新方法及其应用。
我们已经开发了一种快速的方法,以特定位点的方式在肽链内的赖氨酸残基的ε -氨基上结合生物素氨基己丙基部分。利用t-Boc化学固相合成方法和一个碱不稳定、酸稳定的保护基团(Fmoc-)为目标赖氨酸的ε -氨基,我们制备了完全保护的树脂结合肽,这些肽是位点特异性生物素化的。在HF裂解后,获得了纯度高的独特生物素化肽。利用这种方法,制备了许多单环内皮素-1类似物的生物素氨基己丙基衍生物。选择性合成高亲和单环双环类似物,制备了在9位含有epsilon-生物素氨基己丙赖氨酸的双环[Nle7]ET-1类似物。该肽Kd = 0.08 nM,对ETA受体的亲和力比市售的N α生物素化内皮素-1高1000倍。这种生物素化方法的一般效用是通过合成一个位点特异性生物素化的PTH类似物来证明的,该类似物在其序列中包含几个侧链功能化的氨基酸残基。本文报道的合成方法是收敛的,因为它允许间隔物长度的简单变化,并且还提供了以位点特定方式引入其他基团(如亲和标记和荧光标记)的潜力。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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