Trifunctional Probe Building Blocks

Introduction

Chemical probes that mimic a pharmacophore, ligand, or lead compound are of increasing interest for target identification and validation, protein profiling, imaging, or other applications in chemical biology. However, synthesis of the ideal probe is not trivial as even slight structural alterations may have unpredictable effects on target binding and selectivity.¹ The synthesis and screening of several analogs is thus often necessary to optimize the outcome of an assay.

In partnership with Pfizer chemists, we have compiled a collection of trifunctional building blocks to enable development of probes. Each possesses three components:

• A connectivity group – NH₂, CO₂H, OH, CHO, Br, or I – facilitates coupling to ligands.
• A reactive group forms a covalent bond with the protein of interest (POI), allowing even weak-binding events to be captured. Options include diazirines² and benzophenones³ for UV-activated photoaffinity labeling⁴ or sulfonyl fluorides for context-specific labeling.^5,6
• An alkyne bioorthogonal handle permits click-chemistry conjugation of various azide-containing tags for biotin-streptavidin enrichment or fluorescence imaging.

Figure 1. Trifunctional Building Blocks

Advantages

Trifunctional probe building blocks service synthetic chemical biologists in two key ways.

• Reactive groups and bioorthogonal handles are incorporated in one synthetic step.
• Similar connectivity groups enable small library generation.

Applications

These trifunctional scaffolds have been valuable in several applications requiring probe development, such as the examples below.^{1-3, 6-12} To access these probes, a suggested building block is listed next to each structure.

Figure 2. Trifunctional Scaffolds

Library Generation

Similar connectivity groups streamline the functionalization of one bioactive small molecule into a library of potential probes.¹ For instance, by using the CO₂H-containing building blocks with a ligand’s NH₂, a panel of seven probe analogs can be accessed.

Figure 3. Target-specific Probe Library

Product Offering

Figure 4. Benzophenones, Diazirines, and Sulfonyl Fluorides

References

1. Xu H, Hett EC, Gopalsamy A, Parikh MD, Geoghegan KF, Kyne RE, Menard CA, Narayanan A, Robinson RP, Johnson DS, et al. A library approach to rapidly discover photoaffinity probes of the mRNA decapping scavenger enzyme DcpS. Mol. BioSyst.. 11(10):2709-2712. https://doi.org/10.1039/c5mb00288e

2. Li Z, Hao P, Li L, Tan CYJ, Cheng X, Chen GYJ, Sze SK, Shen H, Yao SQ. 2013. Design and Synthesis of Minimalist Terminal Alkyne-Containing Diazirine Photo-Crosslinkers and Their Incorporation into Kinase Inhibitors for Cell- and Tissue-Based Proteome Profiling. Angew. Chem. Int. Ed.. 52(33):8551-8556. https://doi.org/10.1002/anie.201300683

3. Crump CJ, Murrey HE, Ballard TE, am Ende CW, Wu X, Gertsik N, Johnson DS, Li Y. 2016. Development of Sulfonamide Photoaffinity Inhibitors for Probing Cellular ?-Secretase. ACS Chem. Neurosci.. 7(8):1166-1173. https://doi.org/10.1021/acschemneuro.6b00127

4. Murale DP, Hong SC, Haque MM, Lee J. 2016. Photo-affinity labeling (PAL) in chemical proteomics: a handy tool to investigate protein-protein interactions (PPIs). Proteome Sci. 15(1): https://doi.org/10.1186/s12953-017-0123-3

5. Narayanan A, Jones LH. Sulfonyl fluorides as privileged warheads in chemical biology. Chem. Sci.. 6(5):2650-2659. https://doi.org/10.1039/c5sc00408j

6. Fadeyi O, Parikh MD, Chen MZ, Kyne RE, Taylor AP, O'Doherty I, Kaiser SE, Barbas S, Niessen S, Shi M, et al. 2016. Chemoselective Preparation of Clickable Aryl Sulfonyl Fluoride Monomers: A Toolbox of Highly Functionalized Intermediates for Chemical Biology Probe Synthesis. ChemBioChem. 17(20):1925-1930. https://doi.org/10.1002/cbic.201600427

7. Schülke J, McAllister LA, Geoghegan KF, Parikh V, Chappie TA, Verhoest PR, Schmidt CJ, Johnson DS, Brandon NJ. 2014. Chemoproteomics Demonstrates Target Engagement and Exquisite Selectivity of the Clinical Phosphodiesterase 10A Inhibitor MP-10 in Its Native Environment. ACS Chem. Biol.. 9(12):2823-2832. https://doi.org/10.1021/cb500671j

8. Gregory KJ, Velagaleti R, Thal DM, Brady RM, Christopoulos A, Conn PJ, Lapinsky DJ. 2016. Clickable Photoaffinity Ligands for Metabotropic Glutamate Receptor 5 Based on Select Acetylenic Negative Allosteric Modulators. ACS Chem. Biol.. 11(7):1870-1879. https://doi.org/10.1021/acschembio.6b00026

9. Theodoropoulos PC, Gonzales SS, Winterton SE, Rodriguez-Navas C, McKnight JS, Morlock LK, Hanson JM, Cross B, Owen AE, Duan Y, et al. 2016. Discovery of tumor-specific irreversible inhibitors of stearoyl CoA desaturase. Nat Chem Biol. 12(4):218-225. https://doi.org/10.1038/nchembio.2016

10. Xie Y, Ge J, Lei H, Peng B, Zhang H, Wang D, Pan S, Chen G, Chen L, Wang Y, et al. 2016. Fluorescent Probes for Single-Step Detection and Proteomic Profiling of Histone Deacetylases. J. Am. Chem. Soc.. 138(48):15596-15604. https://doi.org/10.1021/jacs.6b07334

11. Li Z, Wang D, Li L, Pan S, Na Z, Tan CYJ, Yao SQ. 2014. Minimalist Cyclopropene-Containing Photo-Cross-Linkers Suitable for Live-Cell Imaging and Affinity-Based Protein Labeling. J. Am. Chem. Soc.. 136(28):9990-9998. https://doi.org/10.1021/ja502780z

12. Zhu B, Zhang H, Pan S, Wang C, Ge J, Lee J, Yao SQ. 2016. In Situ Proteome Profiling and Bioimaging Applications of Small-Molecule Affinity-Based Probes Derived From DOT1L Inhibitors. Chem. Eur. J.. 22(23):7824-7836. https://doi.org/10.1002/chem.201600259