Host–Guest Complexation by β-Cyclodextrin Enhances the Solubility of an Esterified Protein

Abstract

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 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. 1H NMR titration experiments revealed that β-CD encapsulates the hydrophobic tolyl group of ester conjugates with Ka = 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.