Kent S B, Merrifield R B
Int J Pept Protein Res. 1983 Jul;22(1):57-65. doi: 10.1111/j.1399-3011.1983.tb02068.x.
The formation of terminated peptides with free alpha-amino groups has often been observed in stepwise solid phase peptide synthesis. This has been attributed to variable accessibility in regions of the swollen crosslinked resin supports. It is now shown that impurities in the amino acid reagents are responsible for these by-products. Thus, sec.-butyloxycarbonylamino acids were isolated from tert.-butyloxycarbonylamino acids after treatment with trifluoroacetic acid under standard deprotection conditions for the removal of the tert.-butyloxycarbonyl (Boc) group. Direct reverse phase HPLC analysis of Boc-amino acids from commercial sources also showed the sec.-Boc-amino acids as impurities present at varying levels. The sec.-Boc group was stable to treatment at room temperature with trifluoroacetic acid in dichloromethane (1:1, v/v) (half-life 7 years), but was removed by HF-anisole under the standard conditions of cleavage and deprotection of assembled peptides. In model syntheses, the level of terminated free peptides corresponded to the level of preexisting sec.-Boc-amino acid impurities present in the Boc-amino acid reagents. Use of Boc-amino acids with no detectable sec.-Boc resulted in negligible levels (less than 0.05%) of terminated peptides. The problem is thus readily overcome by the use of pure Boc-amino acid starting materials and is not a reflection of a shortcoming inherent to the polymer supported nature of solid phase syntheses as has been previously suggested.
在逐步固相肽合成中，经常会观察到带有游离α-氨基的终止肽的形成。这被归因于交联树脂载体溶胀区域的可及性不同。现在表明，氨基酸试剂中的杂质是这些副产物的原因。因此，在用于去除叔丁氧羰基（Boc）基团的标准脱保护条件下，用三氟乙酸处理后，从叔丁氧羰基氨基酸中分离出仲丁氧羰基氨基酸。对市售Boc氨基酸进行直接反相高效液相色谱分析也表明，仲丁氧羰基氨基酸作为杂质以不同水平存在。仲丁氧羰基在室温下用二氯甲烷（1:1，v/v）中的三氟乙酸处理时是稳定的（半衰期7年），但在组装肽的裂解和脱保护标准条件下可被氢氟酸-苯甲醚去除。在模型合成中，终止游离肽的水平与Boc氨基酸试剂中预先存在的仲丁氧羰基氨基酸杂质水平相对应。使用无可检测到的仲丁氧羰基的Boc氨基酸会导致终止肽的水平可忽略不计（低于0.05%）。因此，通过使用纯的Boc氨基酸起始原料，这个问题很容易得到解决，而且这并不像之前所认为的那样反映了固相合成的聚合物支持性质所固有的缺点。
