An expedient synthesis of highly functionalized 1,3-dienes by employing cyclopropenes as C4 units.

An efficient method has been described to synthesize dicarbonyl functionalized 1,3-dienes by cleaving the C[double bond, length as m-dash]C bond of enaminones with cyclopropenes in the presence of a rhodium catalyst. The acetate-substituted cyclopropenes are judiciously chosen as standard C4 units of 1,3-diene precursors. The reactions are believed to undergo a unique cutting and insertion process, involving a C[double bond, length as m-dash]C bond cleavage of the enaminone and insertion of a new C(sp2) source with the formation of two C-C single bonds. A broad range of substrates can be used to synthesize the corresponding 1,3-dienes under very mild reaction conditions, including low catalyst-loading, ambient temperature, and a neutral reaction solvent.

Genome shuffling based on different types of ribosome engineering mutants for enhanced production of 10-membered enediyne tiancimycin-A.

Tiancimycin-A (TNM-A) is an anthraquinone-fused ten-membered enediyne produced by Streptomyces sp. CB03234, which is very promising for the development of anticancer antibody-drug conjugates (ADCs). To improve the titer of TNM-A, we have generated high-producing mutants CB03234-S and CB03234-R through ribosome engineering, but still not sufficient for pilot production of TNM-A. As the follow-up work, gentamycin-induced ribosome engineering was further adopted here to generate the mutant CB03234-G, which produced similar level of TNM-A as in CB03234-S and CB03234-R. Benefiting from the distinct antibiotic resistances of three ribosome engineering mutants, genome shuffling between any two of them was respectively carried out, and finally obtained the recombinant CB03234-GS26. Under optimal conditions, CB03234-GS26 produced 40.6 ± 1.0 mg/L TNM-A in shaking flasks and 20.8 ± 0.4 mg/L in a scaled-up 30-L fermentor. Comparing with the parental high-producing mutants, the over 1.6-fold titer improvement of CB03234-GS26 in fermentor was more promising for pilot production of TNM-A. Besides the distinctive morphological features, genetic characterization revealed that CB03234-GS26 possessed 1.8 kb rsmG related deletion just the same as CB03234-S, but no mutation was found in rpsL. Subsequent knockouts proved that rsmG was unrelated to titer improvement of TNM-A, which implied other genomic variations and mechanisms rather than ribosome engineering to enhance the biosynthesis of TNM-A. Therefore, CB03234-GS26 provided a basis to locate potential novel genetic targets, and explore the interactions between complex metabolic network and TNM biosynthetic pathway, which shall promote future construction of high-yielding systems for TNM-A and other anthraquinone-fused enediynes.Key Points •United genome shuffling and ribosome engineering help further strain improvement. •CB03234-GS26 with improved titer is practical for the pilot production of TNM-A. •Enhanced TNM-A production should attribute to novel genetic features/mechanisms.

Stereoselective synthesis of trifluoromethyl-substituted 2H-furan-amines from enaminones.

A straightforward strategy for synthesis of highly functionalized trifluoromethyl 2H-furans is described. The copper catalyzed method relies on a cascade cyclic reaction between enaminones and N-tosylhydrazones. This method allows the synthesis of 2-amino-3-trifluoromethyl-substituted 2H-furan derivatives carrying a quaternary stereogenic center as single diastereomers. The proposed reaction mechanism involves an amino-cyclopropane intermediate formed in the cyclopropanation of enaminones. The developed method tolerates a broad spectrum of functionalities, and the obtained 2H-furan derivatives are useful synthetic intermediates for preparing other trifluoromethyl-substituted compounds.

Streptomycin-induced ribosome engineering complemented with fermentation optimization for enhanced production of 10-membered enediynes tiancimycin-A and tiancimycin-D.

Tiancimycins (TNMs) are a group of 10-membered anthraquinone-fused enediynes, newly discovered from Streptomyces sp. CB03234. Among them, TNM-A and TNM-D have exhibited excellent antitumor performances and could be exploited as very promising warheads for the development of anticancer antibody-drug conjugates (ADCs). However, their low titers, especially TNM-D, have severely limited following progress. Therefore, the streptomycin-induced ribosome engineering was adopted in this work for strain improvement of CB03234, and a TNMs high producer S. sp. CB03234-S with the K43N mutation at 30S ribosomal protein S12 was successfully screened out. Subsequent media optimization revealed the essential effects of iodide and copper ion on the production of TNMs, while the substitution of nitrogen source could evidently promote the accumulation of TNM-D, and the ratio of produced TNM-A and TNM-D was responsive to the change of carbon and nitrogen ratio in the medium. Further amelioration of the pH control in scaled up 25 L fermentation increased the average titers of TNM-A and TNM-D up to 13.7 ± 0.3 and 19.2 ± 0.4 mg/L, respectively. The achieved over 45-fold titer improvement of TNM-A, and 109-fold total titer improvement of TNM-A and TNM-D enabled the efficient purification of over 200 mg of each target molecule from 25 L fermentation. Our efforts have demonstrated a practical strategy for titer improvement of anthraquinone-fused enediynes and set up a solid base for the pilot scale production and preclinical studies of TNMs to expedite the future development of anticancer ADC drugs.