Young J D, Huang A S, Ariel N, Bruins J B, Ng D, Stevens R L
Department of Biological Chemistry, UCLA Medical School 90024-1737.
Pept Res. 1990 Jul-Aug;3(4):194-200.
The "classical" Merrifield method was used to synthesize over 500 peptides using Boc-benzyl strategy. The peptides were prepared either manually or on a Beckman 990B synthesizer or an Applied Biosystems 430A synthesizer. Each coupling of Boc amino acid to the growing peptide on the resin was monitored with the ninhydrin reaction. Couplings were considered "incomplete" if there was 99% or less coupling and "high incomplete" if there was 98% or less coupling. The efficiency of coupling was evaluated in regard to the specific amino acids involved in the coupling reaction and to the length of the peptide at the time of the coupling. The most difficult carboxyl-reacting amino acids were histidine, threonine, arginine, valine, isoleucine and glutamine; the most difficult amine reacting residues were glutamine, leucine, alanine, arginine and isoleucine. The number of "incomplete" and "high incomplete" couplings and the total number of monitored couplings of each of the 20 carboxyl-reacting amino acids when reacting with each of the 20 amine-reacting residues were tabulated. Coupling efficiencies decreased with the length of the peptide. The conclusion of this study is that, with the chemistries and methods used in this group of peptides, no amino acid coupling can be predicted to be complete with a single coupling reaction. The study points to the need for on-line determination of coupling efficiency during the synthesis in which a recoupling step is initiated when the first coupling is incomplete.
采用“经典的”梅里菲尔德方法，运用Boc-苄基策略合成了500多种肽。这些肽通过手动方式，或在贝克曼990B合成仪或应用生物系统公司的430A合成仪上制备。用茚三酮反应监测树脂上Boc氨基酸与生长肽的每次偶联。如果偶联率为99%或更低，则认为偶联“不完全”；如果偶联率为98%或更低，则认为偶联“高度不完全”。根据偶联反应中涉及的特定氨基酸以及偶联时肽的长度来评估偶联效率。最难进行羧基反应的氨基酸是组氨酸、苏氨酸、精氨酸、缬氨酸、异亮氨酸和谷氨酰胺；最难进行胺反应的残基是谷氨酰胺、亮氨酸、丙氨酸、精氨酸和异亮氨酸。将20种进行羧基反应的氨基酸与20种进行胺反应的残基反应时，每种氨基酸的“不完全”和“高度不完全”偶联数以及监测到的偶联总数制成表格。偶联效率随肽的长度而降低。本研究的结论是，对于本组肽所使用的化学方法和合成方法，无法预测单次偶联反应能使氨基酸偶联完全。该研究指出在合成过程中需要在线测定偶联效率，当首次偶联不完全时启动重新偶联步骤。
