β-Hydroxy-Stabilized Boron-Nitrogen Heterocycles Enable Rapid and Efficient C-Terminal Protein Modification

Abstract

Bioorthogonal chemistry has enabled the development of bioconjugates in physiological environments while averting interference from endogenous biomolecules. Reactions between carbonyl-containing molecules and alkoxyamines or hydrazines have experienced a resurgence in popularity in bioorthogonal chemistry owing to advances that allow the reactions to occur under physiological conditions. In particular, ortho-carbonyl-substituted phenylboronic acids (CO-PBAs) exhibit greatly accelerated rates of hydrazone and oxime formation via intramolecular Lewis acid catalysis. Unfortunately, the rate of the reverse reaction is also increased, yielding a kinetically less stable bioconjugate. When the substrate is a hydrazine derivative, an intramolecular reaction between the boronic acid and the hydrazone can lead to the formation of a heterocycle containing a boron-nitrogen bond. We have shown previously that α-amino hydrazides undergo rapid reaction with CO-PBAs to form highly stable, tricyclic products, and that this reaction is orthogonal to the popular azide-alkyne and tetrazine-alkene reactions. In this work, we explore a series of heteroatom-substituted hydrazides for their ability to form tricyclic products with two CO-PBAs, 2-formylphenylboronic acid (2fPBA), and 2-acetylphenylboronic acid (AcPBA). In particular, highly stable products were formed using β-hydroxy hydrazides and 2fPBA. C-Terminal β-hydroxy hydrazide proteins are available using conventional biochemical methods, which alleviates one of the difficulties with applications of bioorthogonal chemical reactions: site-specific incorporation of a reactive group into the biomolecular target. Using sortase-mediated ligation (SML), C-terminal threonine and serine hydrazides were appended to a model eGFP protein in high yield. Subsequent labeling with 2fPBA functionalized probes could be performed quickly and quantitatively at neutral pH using micromolar concentrations of reactants. The SML process was applied directly to an expressed protein in cellular extract, and the C-terminal modified target protein was selectively immobilized using 2fPBA-agarose. Elution from the agarose yielded a highly pure protein that retained the hydrazide functionality. This strategy should be generally applicable for rapid, efficient site-specific protein labeling, protein immobilization, and preparation of highly pure functionalized proteins.

Figure. 1

(a) Selected hydrazides bearing various functional groups. (b) Tricyclic DABs generated by reaction between CO-PBAs and 1a - 4c; the boron-heteroatom secondary interactions are labeled in red. (c) Stability of 5a - 8f defined by the percentage of DAB in pH 7 (green) and pH 4 (black) solutions.

Figure. 2

(a) Kinetics of the reactions between 100 μM 4b and 10 μM 2fPBA (black) or AcPBA (red) in 0.1 M phosphate buffer, pH 7. The reaction progress was monitored by the change in absorbance at 302 nm. (b) Equimolar concentrations of 2fPBA and 4b were mixed in pH 7 phosphate buffer to a final concentration of 2 mM or 50 μM. ¹H NMR spectra of the resulting solutions were collected. (c) Crystal structure of 8a.

Figure. 3

(a) Schematic illustration of sortase mediated hydrazinolysis of eGFP-LPETG to yield eGFP-1a which reacted with 2fPBA-TxR or 2fPBA-JF subsequently; hydrolyzed product eGFP-1d was formed in the hydrazinolysis step. (b) Fluorescence (left) and Coomassie blue staining (right) images of a gel which checked the reaction of eGFP-1a and 2fPBA-TxR in pH 7 phosphate buffer or E. coli lysate. The band at ~18 kDa is SrtA. (c) the reactions of eGFP-1a and 2fPBA-JF were monitored by LCMS after 30 minutes (green), 1 hour (orange) and 24 hours (red).

Figure. 4

(a) Schematic illustration of sortase mediated ligation of eGFP-LPETG and 4b/4c to yield eGFP-2a/eGFP-3a which reacted with 2fPBA-JF or 2fPBA-TxR subsequently. (b) LC-MS analysis of sortase mediated ligation of eGFP-LPETG and 4b at 1 hour, 2 hours and 3 hours. (c) Kinetics of conjugation of β-hydroxy hydrazide functionalized eGFP (eGFP-2a) with 2fPBA-JF or AcPBA-JF in pH 7 phosphate buffer. Top panel: eGFP-2a (20 μM) reacted with 2 eq. or 10 eq. of 2fPBA-JF. Bottom panel: kinetics comparison of conjugation of GFP-2a (20 μM) with 10 eq. of 2fPBA-JF or AcPBA-JF. Full LC-MS spectra are shown in Figure S16–18. (d) Fluorescence (left) and Coomassie blue stained (right) images of a gel which demonstrates the labeling of eGFP-2a with 2fPBA-TxR in pH 7 phosphate buffer or E. coli lysate, showing covalent attachment and the orthogonality of the β-hydroxy stabilized DAB conjugation.

Figure. 5

(a) Schematic illustration of protein purification from E. coli cellular lysate. (b) 2fPBA-β-hydroxy hydrazide linkage (8c) reversed by 200 eq. hydrazine in pH 7 buffer. (c) Coomassie blue stained gel which showed successful immobilization of eGFP-2a from impure samples. Lane 1: impure eGFP-2a. Lane 2: eGFP-2a purified by 2fPBA functionalized agarose. Lane 3: cell lysate spiked with eGFP-2a. Lane 4: eGFP-2a purified from cell lysate. (d) Coomassie blue stained gel which showed successful one-pot purification of eGFP from E. coli cellular lysate. Lane 1: E. coli cellular lysate containing eGFP-LPETG. Lane 2: eGFP-2a generated in E. coli cellular lysate. Lane 3: eGFP-2a purified from E. coli cellular lysate.

Scheme. 1

(a) Stepwise representation of tricyclic DAB formation between ortho-carbonyl substituted PBAs and hydrazide derivatives. (b) Fluorescent labeling and immobilization of eGFP through 2fPBA-β-hydroxy hydrazide linkage.