Extended Affinity-guided DMAP Chemistry with a Finely Tuned Acyl Donor for Intracellular FKBP12 Labeling

Abstract

We successfully extended affinity-guided DMAP (AGD) chemistry with a reactivity-tuned acyl donor library that is capable of the selective and efficient labeling of FKBP12 in living cells as well as in test tube. The new AGD chemistry provides great promise for the establishment of selective intracellular protein functionalization or imaging methods.Specific protein labeling with desired functionalities, such as fluorescent probes or purification tags, is a powerful method for the imaging, analysis, or manipulation of natural protein function in living cells.14 One of the most successful techniques is using bioorthogonal reagents and engineered proteins, in which a nonnatural reactive handle (e.g., azide, alkyne, alkene, tetrazine, etc.) or natural peptide/protein tag is genetically introduced. 2,57A part of these approaches permits selective and rapid protein modification with specific reaction even inside the cells, but is inherently limited to label recombinant, nonnatural proteins. Alternatively, an activity-based probe (ABP) is a useful tool for the specific labeling of an "endogenous" enzyme in cells or in vivo. 8,9 However, these probes cannot be applied to label nonenzymatic proteins, and should lead to inactivation of the target enzymes after labeling. Clearly, a universal methodology for the selective and noninvasive chemical labeling of natural proteins is a difficult task and highly desirable.We are now developing ligand-directed (LD) chemistry as a promising strategy for the covalent labeling of endogenous proteins noninvasively. 10 However, "intracellular" protein labeling and imaging is still challenging due to the slow reaction rate of our existing LD chemistry (typically over 12 h incubation is needed).3d,10e In contrast, high-reactive reagents for more rapid protein labeling should cause the nonspecific reaction or decomposition of reagents inside the cells.10c,10f Among our LD chemistry repertories, 4-dimethylaminopyridine (DMAP) tethering a protein ligand exhibited a unique catalytic reaction for selective protein labeling by the proximity effect guided with the specific affinity between a ligand and the target protein (so-called affinity-guided DMAP (AGD) chemistry).11 In AGD chemistry, it was shown that the acyl transfer reaction was greatly facilitated from the thiophenyl ester type of acyl donors to a nucleophilic amino acid residue (such as Lys, Tyr, or Ser) exposed on a target protein by DMAP catalyst (Figure 1a). However, AGD chemistry has not been applicable to intracellular proteins so far, mainly due to the high reactivity of the original acyl donor, while membrane-bound proteins could be efficiently labeled by this chemistry on live cell.11b,11c Thus, we herein sought to finely tune the reactivity of thioesters by varying the leaving group such as 2,4,6-trimethylthiophenol, 3-nitrobenzyl mercaptan (= 3-nitrobenzyl thiol), and 4-chlorobenzyl mercaptan (= 4-chlorobenzyl thiol) (acyl donors 24). The calculated pK a values of these are higher (7.22, 8.97, and 9.32, respectively) tha...

Affinity-Guided Oxime Chemistry for Selective Protein Acylation in Live Tissue Systems

“…As an alternative to transition metals, organocatalysts are also promising for use in protein labeling . We have previously reported an organocatalyst-based protein labeling method, termed affinity-guided 4-dimethylaminopyridine (DMAP) (AGD) chemistry (Figure a) . In this strategy, the affinity ligand–DMAP conjugate selectively binds to the target protein, and the DMAP moiety facilitates an acyl transfer reaction from thiophenyl ester acyl donors containing a reporter tag to the nucleophilic group of an amino acid (e.g., the ε-amino group of Lys and phenol group of Tyr) in the vicinity of the ligand-binding pocket of the POIs.…”

Antagonizing the Androgen Receptor with a Biomimetic Acyltransferase

“…10 They have also used ligand-DMAP conjugates to target intracellular protein targets but with only partial selectivity. 11 …”

Investigating a Boronate‐Affinity‐Guided Acylation Reaction for Labelling Native Antibodies

“…As shown in Figure S3, the selectivity of the acyl transfer reaction was 4.1‐fold for 6 and 10.9‐fold for 7 compared to that of the uncatalysed reaction. The greater selectivity of the latter is attributable to its relatively lower reactivity than that of donor 6 [33] . Moreover, ligand independent nonspecific labelling is minimum, as acyl donor 7 (3.3 μM) alone produced, a similar, an approximately 11‐fold lower signal compared to DMAP‐BA 3 from a triplicate experiment (Figures 2c and S4) and it was employed in all further experiments.…”