Botti P, Ball H L, Lucietto P, Pinori M, Rizzi E, Mascagni P
Department of Peptide Chemistry, Italfarmaco Research Centre, Milan Italy.
J Pept Sci. 1996 Nov-Dec;2(6):371-80. doi: 10.1002/psc.79.
We have previously described the conditions by which peptide synthesis by the solid-phase fragment condensation approach can be carried out using crown ethers as non-covalent protection for the N alpha-amino group. Here we demonstrate that the procedure can be extended to large, partially protected peptide fragments possessing free Lys and/or Arg residues. The first step was to ensure that complex formation on the side chain of amino acids was not detrimental to the methodology and exhibited the same solubility and coupling properties as N alpha-complexed peptides. Thus, a model hexapeptide was synthesized using Fmoc chemistry containing Lys and Arg residues, which, when complexed with 18-Crown-6, was readily soluble in DCM and coupled quantitatively to a resin-bound tetrapeptide. Two tripeptides were then prepared, one containing a free Ser residue, the other free Tyr, to examine the possible occurrence of side reactions. After coupling using standard conditions only the former tripeptide exhibited the formation of the O-acylation by-product (5%). Another model hexapeptide containing Lys, Tyr, Ser and Asp protected with a TFA-stable adamantyl group was complexed with 18-Crown-6 and coupled to the resin-bound tetrapeptide with near quantitative yield. Extending the length of the peptide to 21 and 40 residues, which represent sequences Gly52 to Leu72 (21-mer) and Pro33 to Leu72 (40-mer) from Rattus norvegicus chaperonin 10 protein, respectively, resulted in partially protected fragments that were readily soluble in water, thus enabling purification by RP-HPLC. Complexation with 18-Crown-6 gave two highly soluble products that coupled to resin-board tetramer with 68% and 50% coupling efficiencies for the 21-mer and 40-mer, respectively. Treatment with 1% DIEA solutions followed by acidolytic cleavage and purification of the major product confirmed that the correct product has been formed, when analysed by amino acid analysis and ESI-MS. These results served to extend the methodology of non-covalent protection of large partially protected peptide fragments for the stepwise fragment condensation of polypeptides.
我们之前已经描述了使用冠醚作为Nα-氨基的非共价保护基，通过固相片段缩合方法进行肽合成的条件。在此，我们证明该方法可扩展至具有游离赖氨酸和/或精氨酸残基的大型部分保护的肽片段。第一步是确保氨基酸侧链上的络合物形成对该方法无害，并且表现出与Nα-络合肽相同的溶解性和偶联性质。因此，使用含有赖氨酸和精氨酸残基的Fmoc化学方法合成了一种模型六肽，当与18-冠-6络合时，它很容易溶于二氯甲烷，并定量偶联到树脂结合的四肽上。然后制备了两种三肽，一种含有游离丝氨酸残基，另一种含有游离酪氨酸残基，以研究可能发生的副反应。在使用标准条件偶联后，只有前一种三肽显示出O-酰化副产物的形成（5%）。另一种含有用TFA稳定的金刚烷基保护的赖氨酸、酪氨酸、丝氨酸和天冬氨酸的模型六肽与18-冠-6络合，并以接近定量的产率偶联到树脂结合的四肽上。将肽的长度分别延长至21个和40个残基，它们分别代表来自褐家鼠伴侣蛋白10的Gly52至Leu72（21肽）和Pro33至Leu72（40肽）序列，得到了易溶于水的部分保护片段，从而能够通过反相高效液相色谱进行纯化。与18-冠-6络合得到两种高度可溶的产物，它们与树脂结合的四聚体的偶联效率分别为21肽的68%和40肽的50%。用1%二异丙基乙胺溶液处理，然后进行酸解裂解并纯化主要产物，通过氨基酸分析和电喷雾电离质谱分析证实形成了正确的产物。这些结果有助于扩展用于多肽逐步片段缩合的大型部分保护肽片段的非共价保护方法。
