A Versatile Isocyanate-Mediated Strategy for Appending Chemical Tags onto Drug-Like Small Molecules

Abstract

Target identification studies are a major hurdle in probe and drug discovery pipelines due to the need to chemically modify small molecules of interest, which can be time intensive and have low throughput. Here, we describe a versatile and scalable method for attaching chemical moieties to a small molecule, isocyanate-mediated chemical tagging (IMCT). By preparation of a template resin with an isocyanate capture group and a cleavable linker, nucleophilic groups on small molecules can be modified with an enforced one-to-one stoichiometry. We demonstrate a small molecule substrate scope that includes primary and secondary amines, thiols, phenols, benzyl alcohols, and primary alcohols. Cheminformatic analyses predict that IMCT is reactive with more than 25% of lead-like compounds in publicly available databases. To demonstrate that the method can produce biologically active molecules, we generated FKBP12 photoaffinity labeling (PAL) compounds with a wide range of affinities and showed that purified and crude cleavage products can bind to and label FKBP12. This method could be used to rapidly modify small molecules for many applications, including the synthesis of PAL probes, fluorescence polarization probes, pull-down probes, and degraders.

Scheme 1. (A) Synthesis of Chimeric Molecules with IMCT. (B) Reactivity Screen of Various Nucleophile Groups with IMCT

TEA = triethylamine. AF-DMF = amine free-dimethylformamide. TFA = trifluoroacetic acid. CH₂Cl₂ = dichloromethane. NR = no reaction. * indicates crude yield and HPLC yield were obtained from the same reaction mixture.

Figure 1

(a) Schematic of the PAL synthesis of FKBP12 probes with IMCT. (b) Fluorescent imaging of FKBP12 (green) labeled with PAL probes (10 μM) with or without UV irradiation or rapamycin (10 μM, R) treatment. Alexa Fluor 647 picolyl azide (red) was installed on covalently modified FKBP12 by using CuAAC. ++++ = highest affinity for FKBP12. + = lowest affinity for FKBP12. * indicates crude yield and HPLC yield were obtained from the same reaction mixture.

Figure 2

(a) Fluorescent imaging of FKBP12 (green) labeled with PAL probes (10 μM) with or without parent inhibitor (100 μM) treatment. (b) Fluorescent imaging of FKBP12 (green) labeled with 3 in an 8-point, 5-fold dilution series or linker 7 (10 or 40 μM). All samples were exposed to UV irradiation. Alexa Fluor 647 picolyl azide (red) was installed on covalently modified FKBP12 using CuAAC. ++++ = highest affinity for FKBP12. + = lowest affinity for FKBP12.