The carboxyl groups of a protein can be esterified by reaction with a diazo compound, 2-diazo-2-(p-methylphenyl)-N,N-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 βcyclodextrin (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. 1 H NMR titration experiments revealed that β-CD encapsulates the hydrophobic tolyl group of ester conjugates with K a = 321 M −1 . 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.

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