Yee Hwee Lim

CRISPR-Cas9 strategy for activation of silent Streptomyces biosynthetic gene clusters

Here we report an efficient CRISPR-Cas9 knock-in strategy to activate silent biosynthetic gene clusters (BGCs) in streptomycetes. We applied this one-step strategy to activate multiple BGCs of different classes in five Streptomyces species and triggered the production of unique metabolites, including a novel pentangular type II polyketide in Streptomyces viridochromogenes. This potentially scalable strategy complements existing activation approaches and facilitates discovery efforts to uncover new compounds with interesting bioactivities.

Direct Conversion of Indoles to 3,3-Difluoro-2-oxindoles via Electrophilic Fluorination

3,3-Difluoro-2-oxindoles can be obtained directly from indoles in moderate yields via electrophilic fluorination using N-fluorobenzenesulfonimide as a mild fluorinating reagent. The presence of tert-butyl hydroperoxide during the reaction, together with additional heating after quenching the reaction with triethylamine, is beneficial to the formation of the desired product.

Palladium-meta-Terarylphosphine Catalyst for the Mizoroki–Heck Reaction of (Hetero)Aryl Bromides and Functional Olefins

The evolutionary meta-terarylphosphine ligand architecture of Cy*Phine was recently shown to be a key feature that imposed outstanding performance in palladium-catalyzed copper-free Sonogashira applications. Herein, the Pd-Cy*Phine combination has similarly proven to be a powerful catalyst system for the Mizoroki-Heck reaction. Using high-throughput screening (HTS) methodology, DMF and NaHCO3 were rapidly identified as the most effective solvent and base pair for the cross-coupling catalysis of challenging and industrially valuable substrates including highly electron-rich heteroaryl bromides and unactivated olefins. Unprotected functional groups were well tolerated using low catalyst loadings, and the simple protocol produced excellent yields (up to 99%) with unprecedented substrate diversity. The Pd-Cy*Phine system broadly outperformed many state-of-the-art commercial alternatives, which demonstrated its potential as a next-generation cross-coupling catalyst.

Palladium precatalysts containing meta-terarylphosphine ligands for expedient copper-free Sonogashira cross-coupling reactions

Three novel palladium complexes utilizing different variations of the evolutionary meta-terarylphosphine ligand, Cy*Phine, were developed. These air- and moisture-stable complexes, PdCl2L2 (L = Cy*Phine, Cy*Phine-CF3 and Cy*Phine-nBu), demonstrated exceptional broad-based performance and operational simplicity in the copper-free Sonogashira cross-coupling of challenging (hetero-)aryl chlorides and terminal alkynes. Modifications to the periphery of the ligand scaffold showed modest improvements in the reaction rate when more electron-donating substituents were incorporated, which hints at potential design upgrades in the future.

Decarboxylative/Sonogashira-type cross-coupling using PdCl2(Cy*Phine)2

The PdCl2(Cy*Phine)2 precatalyst containing the meta-terarylphosphine ligand, Cy*Phine, can effectively mediate decarboxylative cross-coupling with a diverse range of (hetero-)aryl, aryl and alkyl chlorides including those with unprotected functionality. Using a facile and robust protocol, this process was extended to the first synthesis of symmetrical di(heteroaryl)alkynes via tandem Sonogashira/decarboxylative cross-coupling of heteroaryl chlorides and propiolic acid.

Auroramycin: A Potent Antibiotic from Streptomyces roseosporus by CRISPR-Cas9 Activation

Silent biosynthetic gene clusters represent a potentially rich source of new bioactive compounds. We report the discovery, characterization, and biosynthesis of a novel doubly glycosylated 24‐membered polyene macrolactam from a silent biosynthetic gene cluster in Streptomyces roseosporus by using the CRISPR‐Cas9 gene cluster activation strategy. Structural characterization of this polyketide, named auroramycin, revealed a rare isobutyrylmalonyl extender unit and a unique pair of amino sugars. Relative and absolute stereochemistry were determined by using a combination of spectroscopic analyses, chemical derivatization, and computational analysis. The activated gene cluster for auroramycin production was also verified by transcriptional analyses and gene deletions. Finally, auroramycin exhibited potent anti‐methicillin‐resistant Staphylococcus aureus (anti‐MRSA) activity towards clinical drug‐resistant isolates.