A Tale of Two Bioconjugations: pH Controlled Divergent Reactivity of Protein α-oxo-Aldehydes in Competing α-oxo-Mannich and Catalyst-Free Aldol Ligations

Abstract

Site-selective chemical methods for protein bioconjugation have revolutionized the fields of cell and chemical biology through the development of novel protein/enzyme probes bearing fluorescent, spectroscopic, or even toxic cargos. Herein, we report two new methods for the bioconjugation of α-oxo aldehyde handles within proteins using small molecule aniline and/or phenol probes. The “α-oxo-Mannich” and “catalyst-free aldol” ligations both compete for the electrophilic α-oxo aldehyde, which displays pH divergent reactivity proceeding through the “Mannich” pathway at acidic pH to afford bifunctionalized bioconjugates, and the “catalyst-free aldol” pathway at neutral pH to afford monofunctionalized bioconjugates. We explore the substrate scope and utility of both of these bioconjugations in the construction of neoglycoproteins, in the process formulating a mechanistic rationale for how both pathways intersect with each other at different reaction pH’s.

Catalyst-free site-selective cross-aldol bioconjugations

“…The discrepancies between the experimentally determined and theoretical m/z values are less than 6 and 3 m/z respectively, and similar degrees of discrepancy are commonly observed during protein mass spectrometry analysis. 20,23,24,27,54 was indeed PSMA-specific via a competition experiment with unlabelled JVZ007 (Fig.…”

“…α-oxo aldehyde JVZ007 was prepared and characterised exactly as previously reported. 24 Azide-JVZ007. To 100 µL of a 50 µM solution of α-oxo aldehyde JVZ007 in pH 7.5 buffer (25 mM sodium phosphate) was added 25 equivalents of 15 (via the delivery of 6.5 µL of a 20 mM solution of 15 in DMSO).…”

“…1,[10][11][12] However, the targeting of canonical amino acids in proteins is seldom site-specific, as multiple copies of the same amino acid may be solvent-exposed. 13 To overcome these limitations, methods have been developed that enable site-specific installation of bioorthogonal functionalities, such as alkynes, [14][15][16][17] azides, 14,15 tetrazines 18,19 and aldehydes, [20][21][22][23][24] into proteins. These bioorthogonal motifs can subsequently be conjugated to, rapidly and selectively.…”

Catalyst-free site-selective cross-aldol bioconjugations

“…We therefore selected the N-terminus for bioconjugation and selectively introduced an α-oxo aldehyde functionality at this site. 20,24,40,48 In order to demonstrate that SCALP bioconjugation could be used in conjunction with existing popular bioconjugation strategies, an azide motif was installed onto α-oxo aldehyde JVZ007 via SCALP with (4-azidophenyl)acetaldehyde 15, affording azide-JVZ007 (Figure 5). Azide-JVZ007 was characterised via protein mass spectrometry and observed in several forms; the expected β-hydroxy aldehyde cross-aldol product, the corresponding hydrate, and hemiacetal (Figure 5).…”

“…1,[10][11][12] However, the targeting of canonical amino acids in proteins is seldom site-specific, as multiple copies of the same amino acid may be solventexposed. 13 To overcome these limitations, methods have been developed that enable site-specific installation of bioorthogonal functionalities, such as alkynes, [14][15][16][17] azides, 14,15 tetrazines 18,19 and aldehydes, [20][21][22][23][24] into proteins. These bioorthogonal motifs can subsequently be conjugated to rapidly and selectively.…”