Methacrylamide, with the molecular formula C?H?NO, has a molecular structure that includes a carbon-carbon double bond and an amide functional group. At room temperature, it is a white crystalline solid. Due to its unsaturated double bond, it can undergo radical polymerization and is commonly used as a copolymer monomer in the production of polymer flocculants, water treatment resins, adhesives, paper strength enhancers, and serves as a fine chemical intermediate in the pharmaceutical and photosensitive materials industries.

Tunable Methacrylamides for Covalent Ligand Directed Release Chemistry

Acrylamides have been widely used as electrophiles for irreversible covalent inhibitors for many proteins bearing noncatalytic cysteines. For example, afatinib, ibrutinib, and AMG-510 are acrylamide-based inhibitors of EGFR, BTK, and K-Ras, respectively. Such irreversible inhibitors have the advantages of nonequilibrium kinetics, full target occupancy, and flexibility to modify the structure for ADME (absorption, distribution, metabolism, and excretion) issues without sacrificing potency and selectivity. Scientists explored α-substituted methacrylamides as electrophilic warheads with varied reactivity, in the context of targeted covalent inhibitors. These compounds form a covalent bond with the nucleophile, which may be followed by the concomitant release of a leaving group that was present at the β-position. Rsearchers showed that substituted methacrylamides in the context of model compounds span a wide window of thiol reactivity (as evaluated by t1/2 for their reaction with GSH), which is predictable and depends on the pKa/b of their respective leaving group. We also showed that these types of electrophiles are suitable for chemoproteomic applications with various proteomic reactivities. As such, these will join a growing collection of cellular-compatible, cysteine-targeting electrophiles that may expand the scope of the targetable cysteinome. Since these methacrylamides leave an identical adduct on proteomically labeled cysteines, mixtures of such compounds may serve in the future as convenient probes for quantitative chemoproteomics with potentially increased coverage.[1]

Here describe α-substituted methacrylamides as a new class of electrophiles suitable for targeted covalent inhibitors. While typically α-substitutions inactivate acrylamides, we show that hetero α-substituted methacrylamides have higher thiol reactivity and undergo a conjugated addition–elimination reaction ultimately releasing the substituent. Their reactivity toward thiols is tunable and correlates with the pKa/pKb of the leaving group. In the context of the BTK inhibitor ibrutinib, these electrophiles showed lower intrinsic thiol reactivity than the unsubstituted ibrutinib acrylamide. This translated to comparable potency in protein labeling, in vitro kinase assays, and functional cellular assays, with improved selectivity. The conjugate addition–elimination reaction upon covalent binding to their target cysteine allows functionalizing α-substituted methacrylamides as turn-on probes. To demonstrate this, we prepared covalent ligand directed release (CoLDR) turn-on fluorescent probes for BTK, EGFR, and K-RasG12C. Rsearchers further demonstrate a BTK CoLDR chemiluminescent probe that enabled a high-throughput screen for BTK inhibitors. Altogether we show that α-substituted methacrylamides represent a new and versatile addition to the toolbox of targeted covalent inhibitor design.

Highly substituted decoupled gelatin methacrylamide

Scientists successfully prepared highly substituted gelatin methacryloyl (GM) and decoupled gelatin methacrylamide (DGM) from GM via removing methacrylate impurities to compare the ambient stability of photocurable functional groups and their cell compatibility. The photocurable methacrylate groups of GM were slowly hydrolyzed into methacrylic acid in neutral solutions at 37 °C whereas the photocurable methacrylamide groups of DGM remained stable even in harsh alkaline and acidic treatments. The photocurable groups in GM and DGM are directly related to the mechanical performance of GM and DGM hydrogels when they are cured by light. Thus, the stability of the photocurable groups in GM and DGM could be an important factor for the quality control of GM and DGM products. GM and DGM exhibited relatively good cell viability. However, soluble byproduct methacrylic acid (MA) lowered cell viability at above 2.5 mg/mL and elicited severe cell toxicity at above 10 mg/mL. Therefore, gelatin methacrylamide with no hydrolysable methacrylate or soluble methacrylic acid impurities could be a safer choice for in vitro or in vivo applications than GM products with hydrolysable methacrylate impurities. In addition, pure photocurable gelatin products without chemical impurities (methacrylate or methacrylic acid) as well as biological impurities (endotoxins) will be highly needed for their translational research in the future.[2]

References

[1]Reddi RN, Resnick E, Rogel A, Rao BV, Gabizon R, Goldenberg K, Gurwicz N, Zaidman D, Plotnikov A, Barr H, Shulman Z, London N. Tunable Methacrylamides for Covalent Ligand Directed Release Chemistry. J Am Chem Soc. 2021 Apr 7;143(13):4979-4992. doi: 10.1021/jacs.0c10644. Epub 2021 Mar 24. PMID: 33761747; PMCID: PMC8041284.

[2]Niu X, Ferracci G, Lin M, Rong X, Zhu M, Cho NJ, Lee BH. Highly substituted decoupled gelatin methacrylamide free of hydrolabile methacrylate impurities: An optimum choice for long-term stability and cytocompatibility. Int J Biol Macromol. 2021 Jan 15;167:479-490. doi: 10.1016/j.ijbiomac.2020.11.187. Epub 2020 Dec 1. PMID: 33275977.