多肽合成经过一百多年的发展，已经达到了一个很高的水平，且通过固相多肽合成仪已实现了自动化。因此，多肽合成经常被大家误认为是已经发展的非常成熟的领域，但其实不然，多肽合成仍然还有很多棘手的问题，特别是肽键形成过程中的消旋副反应一直困扰着该领域的从业者。由于氨基酸具有两个反应位点，二肽合成时需要保护一个氨基酸的氨基和另一个氨基酸的羧基，随后再去除N端或C端保护基，从而再引入第三个氨基酸。化学多肽合成一般采用C到N的延伸方向，并且将单个氨基酸引入肽链需要氨基保护、羧基活化、氨解和脱保护四步。由于化学多肽合成的原子经济性较差，目前最常用的固相多肽合成(SPPS)方法需要使用大量试剂以确保较高的转化率，对环境产生了极大的负面影响。
广州医科大学赵军锋教授团队在2016年，他们首次发现炔酰胺可用作不消旋的多肽缩合剂（J. Am. Chem. Soc., 2016, 138, 13135）。该类缩合试剂结构简单、分子量小（最简单的炔酰胺缩合剂分子量只有133）、稳定性好；反应可以在室温、敞口容器中进行，操作简单、方便；无需任何添加剂和催化剂，极大地体现了其原子经济性的优势。更重要的是含有α-手性中心的羧酸在缩合过程中不会发生消旋，从而提高了目标产物的纯度和收率，降低了产物纯化的难度。
近期赵军锋教授团队在JACS上发表了题为Inverse Peptide Synthesis Using Transient Protected Amino Acids的文章。本文中，作者报道了第一个基于瞬时保护氨基酸的逆向多肽合成方法，通过炔酰胺偶联试剂介导的无外消旋化和差向异构化的实现N到C方向肽链延伸。需要延长的氨基酸的羧基不需要事先保护就可以直接将氨基端进行缩合，利用N,O-双(三甲基硅基)乙酰胺(BSA)可以将氨基酸转化为可溶性甲硅烷酯从而提高其溶解性实现瞬时保护，直接和炔酰胺偶联试剂活化羧酸的中间体反应形成肽键，随后瞬时保护的羧基可以在酸碱处理过程中自发释放，因此可以一锅法实现瞬时保护、活化、氨解和原位脱保护。此方法手性保持率高，收率高，开辟了一条以未保护氨基酸为起始原料的新的多肽合成途径。
作者将氨基保护的氨基酸与炔酰胺试剂MYTsA在二氯甲烷(DCM)中混合反应，随后减压浓缩得到的乙烯基酯中间体无需纯化，便可以在DMF中直接与未保护的氨基酸以及硅烷化试剂BSA反应得到二肽。大多数二肽固体产物无需色谱纯化，只需经过简单的酸碱处理或重结晶即可得到纯二肽产物。
反应操作：
N-Protected amino acid 1 (0.5 mmol), MYTsA 2 (0.55 mmol, 1.1 equiv.) and DCM (3.0 mL)were combined together and stirred until the Nα-Protected amino acid 1 was fully consumed. Afterconcentration under reduced pressure, the corresponding α-acyloxyenamide was obtained, which was directly used in the next step without further purification. Then, a solution of premixed amino acid 3 (1.0 mmol), BSA (1.0 mmol) in DMF (3 mL) were added. The reaction mixture was stirred at room temperature until complete consumption of α-acyloxyenamide.
Purification 1: The reaction mixture was diluted with 0.5 M NaHCO3 (15 mL) and was shook to dissolve the product, the mixture was washed with DCM (10 mL) for three times. Then 2 M HCl was added to the aqueous solution to adjust the pH to 2 (Replacing HCl with 10% citric acid in the presence of tBu or Boc), the aqueous layer was extracted with ethyl acetate (10 mL) for three times.The combined organic layers were washed with 10% citric acid (10 mL) and brine (10 mL), dried over anhydrous MgSO4, filtered, and concentrated under vacuum to offer the dipeptide without further purification.
Purification 2: The reaction mixture was diluted with H2O (15 mL) and added 2 M HCl to adjust the pH to 2 (Replacing HCl with 10% citric acid in the presence of tBu or Boc), the mixture was extracted with ethyl acetate (10 mL) for three times. The combined organic layers were washed with 10% citric acid (10 mL) and brine (10 mL) , dried over anhydrous MgSO4, filtered and concentrated under vacuum. 1 mL ethyl acetate was added to dissolve the residue after concentrated, and 10 mL petroleum ether or diethyl ether was added dropwise and stirred. The precipitate was filtered and dried to afford the pure product.
Purification 3: The reaction mixture was diluted with H2O (15 mL) and added 2 M HCl to adjust the pH to 2 (Replacing HCl with 10% citric acid in the presence of tBu or Boc), the mixture was extracted with ethyl acetate (10 mL) for three times. The combined organic layers were washed with 10% citric acid (10 mL) and brine (10 mL) , dried over anhydrous MgSO4, filtered and concentrated under vacuum. The residue was purified by flash silica-gel chromatography to provide the target product.
反应操作：
N-Protected dipeptidyl acid 5 (0.5mmol), MYTsA 2 (0.55 mmol, 1.1 equiv.) and DCE (5.0 mL) were combined together and stirred until the Nα-Protected dipeptidyl acid 5 was fully consumed. The corresponding α-acyloxyenamide was obtained, which was directly used in the next step without further purification. Then, a solution of premixed amino acid 3 (1.0 mmol), BSA (1.0 mmol) in DCE (5 mL) were added. The reaction mixture was stirred at room temperature until complete consumption of α-acyloxyenamide. The purification method of the Tripeptidyl Acids is the same as that of the Dipeptidyl Acids, which is recrystallized or column chromatography after acidification and extraction.
为进一步证明方法的实用性，作者还研究了该策略在较长肽合成中的应用，结果表明三肽可以很容易延伸为四肽、五肽以及六肽，并且只需简单的酸碱处理和重结晶过程便足以提纯产物。
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