Department of Chemical Engineering, Stanford University, Stanford, California 94305-5025, USA.
Bioconjug Chem. 2010 Feb 17;21(2):255-63. doi: 10.1021/bc9002844.
The tyrosine analog p-propargyloxyphenylalanine (pPa), like tyrosine, has limited water solubility. It has been postulated that this limited solubility has contributed to reduced cellular uptake of pPa and thus reduced in vivo incorporation of pPa into proteins. Using a cell-free protein synthesis system (CFPS) to circumvent cellular uptake, pPa has been incorporated site-specifically into proteins with high specificity at yields up to 27 times greater than the highest previously reported yield. The alkyne group present on proteins incorporated with pPa provides a reactive residue for site-specific bioconjugation with the copper(I)-catalyzed azide-alkyne [3 + 2] cycloaddition (CuAAC). Previously, incorporation of another CuAAC-compatible unnatural amino acid p-azido-l-phenylalanine (pAz) was demonstrated with CFPS. However, incorporation of pPa may be preferred over pAz due to the instability of the pAz's aryl-azido moiety upon UV or near-UV light exposure. Also, the ability to incorporate site-specifically both reactants of the CuAAC (the alkyne group of pPa and the azido group of pAz) into proteins enables direct site-specific conjugation of heterologous proteins. We have demonstrated (for the first time to our knowledge) a one-step, linker-less, site-specific, direct protein-to-protein conjugation using CuAAC and unnatural amino acids.
酪氨酸类似物对丙烯氧基苯丙氨酸（pPa）与酪氨酸一样，水溶性有限。据推测，这种有限的溶解度导致 pPa 的细胞摄取减少，从而导致 pPa 在体内掺入蛋白质的减少。使用无细胞蛋白质合成系统 (CFPS) 绕过细胞摄取，pPa 已被特异性地掺入蛋白质中，特异性高达比以前报道的最高产量高 27 倍。掺入 pPa 的蛋白质上的炔基提供了用于与铜 (I) 催化的叠氮-炔 [3 + 2] 环加成 (CuAAC) 的反应性残基进行位点特异性生物偶联的反应性残基。以前，已经使用 CFPS 证明了另一种与 CuAAC 兼容的非天然氨基酸 p-叠氮基-l-苯丙氨酸 (pAz) 的掺入。然而，由于 pAz 的芳基叠氮部分在 UV 或近 UV 光暴露下的不稳定性，pPa 的掺入可能优于 pAz。此外，能够特异性地将 CuAAC 的两种反应物（pPa 的炔基和 pAz 的叠氮基）掺入蛋白质中，能够直接进行特异性的蛋白质-蛋白质偶联。我们已经首次（据我们所知）使用 CuAAC 和非天然氨基酸证明了一步、无接头、特异性、直接的蛋白质-蛋白质偶联。
