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 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.

Modular Diazo Compound for the Bioreversible Late-Stage Modification of Proteins

“…31 Such extensive modification can compromise conformational stability and aqueous solubility of a protein. 33 To enable protein delivery with fewer pendant esters, we designed a diazo compound that enables the addition of highly potent delivery domains. Specifically, we supplied diazo compound 1 with a pyridyl disulfide, which allows for rapid thiol−disulfide interchange under mild conditions.…”

“…Recently, we reported on α-aryl-α-diazoacetamidesa new class of bioreversible modification reagents that O -alkylate protein carboxyl groups chemoselectively under physiological conditions. − Such esterification makes the protein surface more cationic and hydrophobic, thereby enabling the delivery of proteins into the cytosol. , Still, the successful delivery of GFP, which is an anionic protein with Z = −9 (where Z refers to Arg + Lys – Asp – Glu), requires the esterification of a dozen or so carboxyl groups . Such extensive modification can compromise conformational stability and aqueous solubility of a protein …”

Evaluation of the Cytosolic Uptake of HaloTag Using a pH-Sensitive Dye

“…The identity of the conjugates was confirmed using mass spectrometry and found to contain either 0–3 labels (low labeling) or 2–8 (high labeling) (Figures S7 and S8). Installation of more labels on the surface of HaloTag led to the precipitation of the protein, consistent with previous reports correlating the number of labels with protein insolubility as the p I of the protein approaches the pH of the solution …”

“…Installation of more labels on the surface of HaloTag led to the precipitation of the protein, consistent with previous reports correlating the number of labels with protein insolubility as the pI of the protein approaches the pH of the solution. 78 Recently, we deployed a novel diazo compound, 2-SSpy, that enables a late-stage modification with a ligand of choice. 36 To modify HaloTag with this compound, 2-SSpy was briefly premixed with the thiol-equipped R10 to form a disulfide linkage prior to reaction with the protein.…”

Toward a Traceless Tag for the Thiol‐Mediated Uptake of Proteins

“…GFP 8 was selected as the initial model POI. It soon became apparent that esterification under the original conditions gave low yields [59] of conjugate 9 , mainly due to the poor solubility of the product. This outcome could be attributed to the hydrophobic nature of the ester and the removal of the negative charges resulting in an increasing isoelectric point toward isoelectric precipitation under physiological conditions [60–62] …”