Mol Pharm. 2022 Nov 7;19(11):3869-3876. doi: 10.1021/acs.molpharmaceut.2c00368. Epub 2022 Aug 29.
The carboxyl groups of a protein can be esterified by reaction with a diazo compound, 2-diazo-2-(-methylphenyl)-,-dimethylacetamide. This esterification enables the entry of the protein into the cytosol of a mammalian cell, where the nascent ester groups are hydrolyzed by endogenous esterases. The low aqueous solubility of the ensuing esterified protein is, however, a major practical challenge. Solubility screening revealed that β-cyclodextrin (β-CD) is an optimal solubilizing agent for esterified green fluorescent protein (est-GFP). Its addition can increase the recovery of est-GFP by 10-fold. α-CD, γ-CD, and cucurbit-7-uril are less effective excipients. H NMR titration experiments revealed that β-CD encapsulates the hydrophobic tolyl group of ester conjugates with = 321 M. Combining l-arginine and sucrose with β-CD enables the nearly quantitative recovery of est-GFP. Thus, the insolubility of esterified proteins can be overcome with excipients.
蛋白质的羧基可以与重氮化合物 2-二氮-2-(-甲基苯基)-,-二甲基乙酰胺反应而酯化。这种酯化作用使蛋白质进入哺乳动物细胞的细胞质，其中新生的酯基被内源性酯酶水解。然而，随后酯化的蛋白质的低水溶解度是一个主要的实际挑战。溶解度筛选表明β-环糊精（β-CD）是酯化绿色荧光蛋白（est-GFP）的最佳增溶剂。它的添加可以使 est-GFP 的回收率提高 10 倍。α-CD、γ-CD 和瓜环-7-脲的效果较差。1 H NMR 滴定实验表明，β-CD 用 = 321 M 包封了酯结合物的疏水性甲苯基。将 l-精氨酸和蔗糖与 β-CD 结合使用，可以使 est-GFP 的回收率接近定量。因此，可以用赋形剂克服酯化蛋白的不溶性。
