Catalyst-mediated protein modification is a powerful approach for the imaging and engineering of natural proteins. We have previously developed affinity-guided 4-dimethylaminopyridine (AGD) chemistry as an efficient protein modification method using a catalytic acyl transfer reaction. However, because of the high electrophilicity of the thioester acyl donor molecule, AGD chemistry suffers from nonspecific reactions to proteins other than the target protein in crude biological environments, such as cell lysates, live cells, and tissue samples. To overcome this shortcoming, we here report a new acyl donor/organocatalyst system that allows more specific and efficient protein modification. In this method, a highly nucleophilic pyridinium oxime (PyOx) catalyst is conjugated to a ligand specific to the target protein. The ligand-tethered PyOx selectively binds to the target protein and facilitates the acyl transfer reaction of a mild electrophilic N-acyl-N-alkylsulfonamide acyl donor on the protein surface. We demonstrated that the new catalytic system, called AGOX (affinity-guided oxime) chemistry, can modify target proteins, both in test tubes and cell lysates, more selectively and efficiently than AGD chemistry. Low-background fluorescence labeling of the endogenous cell-membrane proteins, carbonic anhydrase XII and the folate receptor, in live cells allowed for the precise quantification of diffusion coefficients in the protein's native environment. Furthermore, the excellent biocompatibility and bioorthogonality of AGOX chemistry were demonstrated by the selective labeling of an endogenous neurotransmitter receptor in mouse brain slices, which are highly complicated tissue samples.

Glycoproteins on cell surfaces play important roles in biological processes, including cell-cell interaction/signaling, immune response, and cell differentiation. Given the diversity of the structure of glycans, labeling and imaging of selected glycoproteins are challenging, although several promising strategies have been developed recently. Here, we design and construct semisynthetic reactive lectins (sugar-binding proteins) that are able to selectively label glycoproteins. Congerin II, an animal galectin, and wheat germ agglutinin are conjugated with 4-dimethylaminopyridine (DMAP), a well-known acyl transfer catalyst by our affinity-guided DMAP method and Cu(I)-assisted click chemistry. Selective labeling of glycoproteins is facilitated by the DMAP-tethered lectin catalysts both in vitro and on living cells. Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) analysis enabled us to isolate labeled glycoproteins that are uniquely exposed on distinct cell lines. Furthermore, the combination of immunoprecipitation with mass spectrometry (MS)-fingerprinting techniques allowed us to characterize 48 glycoproteins endogenously expressed on HeLa cells, and some low-abundant glycoproteins, such as epidermal growth factor receptor (EGFR) and neuropilin-1, were successfully identified. Our results demonstrate that semisynthetic DMAP-tethered lectins provide a new tool for labeling and profiling glycoproteins on living cells.

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...