Contemporary chemical protein synthesis has been dramatically advanced over the past few decades, which has enabled chemists to reach the landscape of synthetic biomacromolecules. Chemical synthesis can produce synthetic proteins with precisely controlled structures which are difficult or impossible to obtain via gene expression systems. Herein, we summarize the key enabling ligation technologies, major strategic developments, and some selected representative applications of synthetic proteins and provide an outlook for future development.

Trimethylsilyl (TMS) groups present outstanding NMR probes of biological macromolecules as they produce intense singlets in 1H NMR spectra near 0 ppm, where few other proton resonances occur. We report a system for genetic encoding of N 6-(((trimethylsilyl)­methoxy)­carbonyl)-l-lysine (TMSK) for site-specific incorporation into proteins. The system is based on pyrrolysyl-tRNA synthetase mutants, which deliver proteins with high yield and purity in vivo and in cell-free protein synthesis. As the TMS signal can readily be identified in 1D 1H NMR spectra of high-molecular weight systems without the need of isotopic labeling, TMSK delivers an excellent site-specific NMR probe for the study of protein structure and function, which is both inexpensive and convenient. We demonstrate the utility of TMSK to detect ligand binding, measure the rate of conformational change, and assess protein dimerization by paramagnetic relaxation enhancement. In addition, we present a system for dual incorporation of two different unnatural amino acids (TMSK and O-tert-butyl-tyrosine) in the same protein in quantities sufficient for NMR spectroscopy. Close proximity of the TMS and tert-butyl groups was readily detected by nuclear Overhauser effects.

Analysis of the whole genome sequence of Streptomyces sp. IMB7-145 revealed the presence of seven type I polyketide synthase biosynthetic gene clusters, one of which was highly homologous to the biosynthetic gene cluster of azalomycin F. Detailed bioinformatic analysis of the modular organization of the PKS gene suggested that this gene is responsible for niphimycin biosynthesis. Guided by genomic analysis, a large-scale cultivation ultimately led to the discovery and characterization of four new niphimycin congeners, namely, niphimycins C-E (1-3) and 17-O-methylniphimycin (4). The configurations of most stereocenters of niphimycins have not been determined to date. In the present study, the relative configurations were elucidated by spectroscopic analysis, including J-based analysis and the CNMR database method. Further, the full absolute configurations of niphimycins were completely proposed for the first time based on biosynthetic gene cluster analysis of the ketoreductase and enoylreductase domains for hydroxy- and methyl-bearing stereocenters. Compounds 1, 3, 4, and niphimycin Iα (5) showed antimicrobial activity against methicillin-resistant Staphylococcus aureus and vancomycin-resistant enterococci (MIC: 8-64 μg/mL), as well as cytotoxicity against the human HeLa cancer cell line (IC: 3.0-9.0 μM). In addition, compounds 1 and 5 displayed significant activity against several Mycobacterium tuberculosis clinical isolates (MIC: 4-32 μg/mL).