Xiaojing Liu, Jun Yang, Lei Gao, Liyun Zhang, and Xiaoguang Lei
Diels–Alder reaction is one of the most important transformations used in organic synthesis, with the ability to construct two new C–C bonds and up to four chiral centers simultaneously. However, the biggest synthetic challenge in Diels–Alder reaction lies in controlling its regio-, diastereo-, and enantioselectivity.Using Stille cross-coupling and enzymatic Diels–Alder reaction as the key steps, the first chemoenzymatic total synthesis of artonin I is achieved in 30% overall yield over only seven steps. This enzymatic Diels–Alder reaction catalyzed by MaDA is featured with excellent endo- and enantioselectivity and high catalytic efficiency (kcat/KM = 362 ± 54 mm−1 s−1). These successful chemoenzymatic total syntheses of artonin I and dideoxyartonin I demonstrated the remarkable potential of the intermolecular Diels–Alderase MaDA in biocatalysis.
Weilong Liu, Zongwei Yue, Zhen Wang, Houhua Li, and Xiaoguang Lei
We report herein the diversity-oriented synthesis of various tetracyclic Isodon diterpenoid scaffolds guided by radical cyclization logic. Our substrate-based dienyne radical cyclization approach is distinctive from reagent-based rearrangement approaches that are generally applied in biosynthesis or previous synthetic studies. An unprecedented cyclization at C14 via 1,5- radical translocation/5-exo-trig cyclization is observed, which enriches our radical cyclization pattern. Furthermore, biological evaluations revealed that several new natural product-like compounds showed promising anticancer activities against various cancer cell lines.
Lei Gao, Cong Su, Xiaoxia Du, Ruishan Wang, Shuming Chen, Yu Zhou, Chengwei Liu, Xiaojing Liu, Runze Tian, Liyun Zhang, Kebo Xie, She Chen, Qianqian Guo, Lanping Guo, Yoshio Hano, Manabu Shimazaki , Atsushi Minami, Hideaki Oikawa, Niu Huang, K. N. Houk, Luqi Huang*, Jungui Dai* and Xiaoguang Lei*
Nature Chem. 2020, https://doi.org/10.1038/s41557-020-0467-7
The Diels–Alder reaction is one of the most powerful and widely used methods in synthetic chemistry for the stereospecific construction of carbon–carbon bonds. Despite the importance of Diels–Alder reactions in the biosynthesis of numerous secondary metabolites, no naturally occurring stand-alone Diels–Alderase has been demonstrated to catalyse intermolecular Diels– Alder transformations. Here we report a flavin adenine dinucleotide-dependent enzyme, Morus alba Diels–Alderase (MaDA), from Morus cell cultures, that catalyses an intermolecular [4+2] cycloaddition to produce the natural isoprenylated flavonoid chalcomoracin with a high efficiency and enantioselectivity. Density functional theory calculations and preliminary measurements of the kinetic isotope effects establish a concerted but asynchronous pericyclic pathway. Structure-guided mutagenesis and docking studies demonstrate the interactions of MaDA with the diene and dienophile to catalyse the [4+2] cycloaddition. MaDA exhibits a substrate promiscuity towards both dienes and dienophiles, which enables the expedient syntheses of structurally diverse natural products. We also report a biosynthetic intermediate probe (BIP)-based target identification strategy used to discover MaDA.
The β-lactam antibiotic resistance has become a critical global health threat. One of the major reasons for drug resistance is the expression of β-lactamases especially metallo-β-lactamases such as New Delhi metallo-β-lactamase (NDM-1) by Gram-negative bacteria. The fungal natural product aspergillomarasmine A (AMA) was found to be a promising inhibitor of NDM-1 to potentiate currently used β-lactam antibiotics to overcome drug resistance both in vitro and in vivo. Although several chemical synthesis and chemoenzymatic synthesis approaches to access AMA have been reported, the biosynthesis of AMA was still elusive. Herein, we identified the key enzyme responsible for the biosynthesis of AMA in Aspergillus oryzae. AMA synthase is a PLP-dependent cysteine synthase homologous protein which utilizes O-acetyl-Lserine/O-phospho-L-serine and L-aspartic acid as its substrates. Remarkably, this enzyme catalyzes two consecutive C-N bond formations to produce AMA efficiently which may be attributed to the spacious substrate-binding pocket. PLP is covalently bound to Lys61 by an internal aldimine from the PLP re face, and the si face of PLP pyridine ring is accessible to the substrates to promote the nucleophilic addition of amino acids to the double bond of the external adiminine and ultimately to generate chiral Cα with S configuration. The catalytic mechanism was proposed based on molecular docking and biochemical experiments. In addition, we have further investigated the substrate scope of AMA synthase and identified a variant enzyme which shows promising potential in producing structurally diverse molecules containing C-N bond.
Tuberculosis (TB) is a life-threatening disease resulting in an estimated 10 million new infections and 1.8 million deaths annually, primarily in underdeveloped countries. The economic burden of TB has been estimated as approximately 12 billion USD annually in direct and indirect costs. Additionally, multi-drug-resistant (MDR) and extreme-drug-resistant (XTR) TB strains resulting in about 250 000 deaths annually are now widespread, increasing pressure on the identification of new anti-TB agents that operate by a novel mechanism of action. Chrysomycin A is a rare C-aryl glycoside first discovered over 60 years ago. In a recent highthroughput screen, we found that chrysomycin A has potent anti-TB activity, with minimum inhibitory concentration (MIC) = 0.4 μg/mL against MDR-TB strains. However, chrysomycin A is obtained in low yields from fermentation of Streptomyces, and the mechanism of action of this compound is unknown. To facilitate the mechanism of action and preclinical studies of chrysomycin A, we developed a 10-step, scalable synthesis of the isolate and its two natural congeners polycarcin V and gilvocarcin V. The synthetic sequence was enabled by the implementation of two sequential C−H functionalization steps as well as a late-stage C-glycosylation. In addition, >10 g of the advanced synthetic intermediate has been prepared, which greatly facilitated the synthesis of 33 new analogues to date. The structure−activity relationship was subsequently delineated, leading to the identification of derivatives with superior potency against MDR-TB (MIC = 0.08 μg/mL). The more potent derivatives contained a modified carbohydrate residue which suggests that further optimization is additionally possible. The chemistry we report here establishes a platform for the development of a novel class of anti-TB agents active against drug-resistant pathogens.
Late-stage diversification of natural products is an efficient way to generate natural product derivatives for drug discovery and chemical biology. Benefiting from the development of site-selective synthetic methodologies, late-stage diversification of natural products has achieved notable success. This outlook will outline selected examples of novel methodologies for site-selective transformations of reactive functional groups and inert C–H bonds that enable late-stage diversification of complex natural products. Accordingly, late-stage diversification provides an opportunity to rapidly access various derivatives for modifying lead compounds, identifying cellular targets, probing protein–protein interactions, and elucidating natural product biosynthetic relationships.
Yingjie Bai, Hiu C. Lam, and Xiaoguang Lei*
Acc. Chem. Rev. 2020, https://doi.org/10.1021/acs.accounts.9b00600
we developed a biomimetic synthetic strategy based on diverse Diels–Alder reactions in the total syntheses of ainsliadimers A and B, ainsliatrimers A and B, and gonchnatiolides A–C, which are natural product inhibitors or activators for PCD. Using synthetic ainsliadimer A probe, we elucidated that ainsliadimer A inhibits the NF-κB pathway by covalently binding to Cys46 of IKKβ and triggers apoptosis of cancer cells. We have also revealed that IKKβ is allosterically inhibited by ainsliadimer A. In addition to total synthesis, we have developed a bioorthogonal click hetero-Diels–Alder cycloaddition of vinyl thioether and o-quinolinone quinone methide (TQ-ligation) to facilitate small molecule target identification. The combination of total synthesis and TQ-ligation enables subcellular imaging and identification of the cellular target of ainsliatrimer A to be PPARγ. In addition, TQ-ligation has been applied in the discovery of heat shock protein 90 (HSP90) as one of the functional target proteins for kongensin A. We also confirmed that kongensin A covalently attaches to Cys420 within HSP90 and demonstrated that kongensin A blocks the interaction between HSP90 and CDC37 and subsequently inhibits necroptosis. Our development of these diverse PCD modulators provides not only effective chemical tools for fundamental biomedical research, but also the foundation for drug discovery targeting important human diseases such as cancers and inflammation caused by malfunction of PCD.
Plants deploy a variety of secondary metabolites to fend off pathogen attack. Although defense compounds are generally considered toxic to microbes, the exact mechanisms are often unknown. Here, we show that the Arabidopsis defense compound sulforaphane (SFN) functions primarily by inhibiting Pseudomonas syringae type III secretion system (TTSS) genes, which are essential for pathogenesis. Plants lacking the aliphatic glucosinolate pathway, which do not accumulate SFN, were unable to attenuate TTSS gene expression and exhibited increased susceptibility to P. syringae strains that cannot detoxify SFN. Chemoproteomics analyses showed that SFN covalently modified the cysteine at position 209 of HrpS, a key transcription factor controlling TTSS gene expression. Site-directed mutagenesis and functional analyses further confirmed that Cys209 was responsible for bacterial sensitivity to SFN in vitro and sensitivity to plant defenses conferred by the aliphatic glucosinolate pathway. Collectively, these results illustrate a previously unknown mechanism by which plants disarma pathogenic bacterium
Xiaoyun Zhang, Shuo-Qing Zhang, Qiang Li, Fan Xiao, Zongwei Yue, Xin Hong,* and Xiaoguang Lei*
Org. Lett. 2020, 22, 8, 2920-2924
We report here a deep mechanistic study of the “click” ortho-quinone methide (oQM) cycloaddition between orthoquinolinone quinone methide (oQQM) and thio-vinyl ether (TV), named as TQ-ligation. DFT calculations revealed the unexpected fact that dehydration of oQQM precursors is the rate-determining step of this transformation, and two highly reactive oQQM precursors were predicted. Guided by the calculations, a new “click” oQM cycloaddition which shows significantly improved kinetics and remarkable efficiency on protein labeling was developed.
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Chemoenzymatic Total Syntheses of Artonin I with an Intermolecular Diels–Alderase
Xiaojing Liu, Jun Yang, Lei Gao, Liyun Zhang, and Xiaoguang Lei
Diels–Alder reaction is one of the most important transformations used in organic synthesis, with the ability to construct two new C–C bonds and up to four chiral centers simultaneously. However, the biggest synthetic challenge in Diels–Alder reaction lies in controlling its regio-, diastereo-, and enantioselectivity.Using Stille cross-coupling and enzymatic Diels–Alder reaction as the key steps, the first chemoenzymatic total synthesis of artonin I is achieved in 30% overall yield over only seven steps. This enzymatic Diels–Alder reaction catalyzed by MaDA is featured with excellent endo- and enantioselectivity and high catalytic efficiency (kcat/KM = 362 ± 54 mm−1 s−1). These successful chemoenzymatic total syntheses of artonin I and dideoxyartonin I demonstrated the remarkable potential of the intermolecular Diels–Alderase MaDA in biocatalysis.
Syntheses of Skeletally Diverse Tetracyclic Isodon Diterpenoid Scaffolds Guided by Dienyne Radical Cyclization Logic
Weilong Liu, Zongwei Yue, Zhen Wang, Houhua Li, and Xiaoguang Lei
We report herein the diversity-oriented synthesis of various tetracyclic Isodon diterpenoid scaffolds guided by radical cyclization logic. Our substrate-based dienyne radical cyclization approach is distinctive from reagent-based rearrangement approaches that are generally applied in biosynthesis or previous synthetic studies. An unprecedented cyclization at C14 via 1,5- radical translocation/5-exo-trig cyclization is observed, which enriches our radical cyclization pattern. Furthermore, biological evaluations revealed that several new natural product-like compounds showed promising anticancer activities against various cancer cell lines.
Chemical screening identifies ROCK1 as a regulator of migrasome formation
Puzhong Lu, Rui Liu, Di Lu, Yue Xu, Xueyi Yang, Zheng Jiang, Chun Yang, Li Yu*, Xiaoguang Lei* & Yang Chen*
Cell Discovery 2020, 6, 51
FAD-dependent enzyme-catalysed intermolecular [4+2] cycloaddition in natural product biosynthesis
Lei Gao, Cong Su, Xiaoxia Du, Ruishan Wang, Shuming Chen, Yu Zhou, Chengwei Liu, Xiaojing Liu, Runze Tian, Liyun Zhang, Kebo Xie, She Chen, Qianqian Guo, Lanping Guo, Yoshio Hano, Manabu Shimazaki , Atsushi Minami, Hideaki Oikawa, Niu Huang, K. N. Houk, Luqi Huang*, Jungui Dai* and Xiaoguang Lei*
Nature Chem. 2020, https://doi.org/10.1038/s41557-020-0467-7
The Diels–Alder reaction is one of the most powerful and widely used methods in synthetic chemistry for the stereospecific construction of carbon–carbon bonds. Despite the importance of Diels–Alder reactions in the biosynthesis of numerous secondary metabolites, no naturally occurring stand-alone Diels–Alderase has been demonstrated to catalyse intermolecular Diels– Alder transformations. Here we report a flavin adenine dinucleotide-dependent enzyme, Morus alba Diels–Alderase (MaDA), from Morus cell cultures, that catalyses an intermolecular [4+2] cycloaddition to produce the natural isoprenylated flavonoid chalcomoracin with a high efficiency and enantioselectivity. Density functional theory calculations and preliminary measurements of the kinetic isotope effects establish a concerted but asynchronous pericyclic pathway. Structure-guided mutagenesis and docking studies demonstrate the interactions of MaDA with the diene and dienophile to catalyse the [4+2] cycloaddition. MaDA exhibits a substrate promiscuity towards both dienes and dienophiles, which enables the expedient syntheses of structurally diverse natural products. We also report a biosynthetic intermediate probe (BIP)-based target identification strategy used to discover MaDA.
Identification of the AMA Synthase from the Aspergillomarasmine A Biosynthesis and Evaluation of Its Biocatalytic Potential
Qianqian Guo, Dongshan Wu, Lei Gao, Yingjie Bai, Yuan Liu, Nianxin Guo, Xiaoxia Du, Jun Yang, Xiaoming Wang, and Xiaoguang Lei
ACS Catal. 2020, https://doi/10.1021/acscatal.0c01187
The β-lactam antibiotic resistance has become a critical global health threat. One of the major reasons for drug resistance is the expression of β-lactamases especially metallo-β-lactamases such as New Delhi metallo-β-lactamase (NDM-1) by Gram-negative bacteria. The fungal natural product aspergillomarasmine A (AMA) was found to be a promising inhibitor of NDM-1 to potentiate currently used β-lactam antibiotics to overcome drug resistance both in vitro and in vivo. Although several chemical synthesis and chemoenzymatic synthesis approaches to access AMA have been reported, the biosynthesis of AMA was still elusive. Herein, we identified the key enzyme responsible for the biosynthesis of AMA in Aspergillus oryzae. AMA synthase is a PLP-dependent cysteine synthase homologous protein which utilizes O-acetyl-Lserine/O-phospho-L-serine and L-aspartic acid as its substrates. Remarkably, this enzyme catalyzes two consecutive C-N bond formations to produce AMA efficiently which may be attributed to the spacious substrate-binding pocket. PLP is covalently bound to Lys61 by an internal aldimine from the PLP re face, and the si face of PLP pyridine ring is accessible to the substrates to promote the nucleophilic addition of amino acids to the double bond of the external adiminine and ultimately to generate chiral Cα with S configuration. The catalytic mechanism was proposed based on molecular docking and biochemical experiments. In addition, we have further investigated the substrate scope of AMA synthase and identified a variant enzyme which shows promising potential in producing structurally diverse molecules containing C-N bond.
Chrysomycin A Derivatives for the Treatment of Multi-Drug- Resistant Tuberculosis
Fan Wu, Jing Zhang, Fuhang Song, Sanshan Wang, Hui Guo, Qi Wei, Huanqin Dai, Xiangyin Chen, Xuekui Xia, Xueting Liu, Lixin Zhang, Jin-Quan Yu, and Xiaoguang Lei*
ACS Cent. Sci. 2020, https://dx.doi.org/10.1021/acscentsci.0c00122
Tuberculosis (TB) is a life-threatening disease resulting in an estimated 10 million new infections and 1.8 million deaths annually, primarily in underdeveloped countries. The economic burden of TB has been estimated as approximately 12 billion USD annually in direct and indirect costs. Additionally, multi-drug-resistant (MDR) and extreme-drug-resistant (XTR) TB strains resulting in about 250 000 deaths annually are now widespread, increasing pressure on the identification of new anti-TB agents that operate by a novel mechanism of action. Chrysomycin A is a rare C-aryl glycoside first discovered over 60 years ago. In a recent highthroughput screen, we found that chrysomycin A has potent anti-TB activity, with minimum inhibitory concentration (MIC) = 0.4
μg/mL against MDR-TB strains. However, chrysomycin A is obtained in low yields from fermentation of Streptomyces, and the mechanism of action of this compound is unknown. To facilitate the mechanism of action and preclinical studies of chrysomycin A, we developed a 10-step, scalable synthesis of the isolate and its two natural congeners polycarcin V and gilvocarcin V. The synthetic
sequence was enabled by the implementation of two sequential C−H functionalization steps as well as a late-stage C-glycosylation. In addition, >10 g of the advanced synthetic intermediate has been prepared, which greatly facilitated the synthesis of 33 new analogues to date. The structure−activity relationship was subsequently delineated, leading to the identification of derivatives with
superior potency against MDR-TB (MIC = 0.08 μg/mL). The more potent derivatives contained a modified carbohydrate residue which suggests that further optimization is additionally possible. The chemistry we report here establishes a platform for the development of a novel class of anti-TB agents active against drug-resistant pathogens.
Late-Stage Diversification of Natural Products
Benke Hong, Tuoping Luo, Xiaoguang Lei*
ACS Cent. Sci. (Cover Story) https://doi.org/10.1021/acscentsci.9b00916
Late-stage diversification of natural products is an efficient way to generate natural product derivatives for drug discovery and chemical biology. Benefiting from the development of site-selective synthetic methodologies, late-stage diversification of natural products has achieved notable success. This outlook will outline selected examples of novel methodologies for site-selective transformations of reactive functional groups and inert C–H bonds that enable late-stage diversification of complex natural products. Accordingly, late-stage diversification provides an opportunity to rapidly access various derivatives for modifying lead compounds, identifying cellular targets, probing protein–protein interactions, and elucidating natural product biosynthetic relationships.
Dissecting Programmed Cell Death with Small Molecules
Yingjie Bai, Hiu C. Lam, and Xiaoguang Lei*
Acc. Chem. Rev. 2020, https://doi.org/10.1021/acs.accounts.9b00600
we developed a biomimetic synthetic strategy based on diverse Diels–Alder reactions in the total syntheses of ainsliadimers A and B, ainsliatrimers A and B, and gonchnatiolides A–C, which are natural product inhibitors or activators for PCD. Using synthetic ainsliadimer A probe, we elucidated that ainsliadimer A inhibits the NF-κB pathway by covalently binding to Cys46 of IKKβ and triggers apoptosis of cancer cells. We have also revealed that IKKβ is allosterically inhibited by ainsliadimer A. In addition to total synthesis, we have developed a bioorthogonal click hetero-Diels–Alder cycloaddition of vinyl thioether and o-quinolinone quinone methide (TQ-ligation) to facilitate small molecule target identification. The combination of total synthesis and TQ-ligation enables subcellular imaging and identification of the cellular target of ainsliatrimer A to be PPARγ. In addition, TQ-ligation has been applied in the discovery of heat shock protein 90 (HSP90) as one of the functional target proteins for kongensin A. We also confirmed that kongensin A covalently attaches to Cys420 within HSP90 and demonstrated that kongensin A blocks the interaction between HSP90 and CDC37 and subsequently inhibits necroptosis. Our development of these diverse PCD modulators provides not only effective chemical tools for fundamental biomedical research, but also the foundation for drug discovery targeting important human diseases such as cancers and inflammation caused by malfunction of PCD.
An Arabidopsis Secondary Metabolite Directly Targets Expression of the Bacterial Type III Secretion System to Inhibit Bacterial Virulence
Wei Wang, Jing Yang, Jian Zhang, Yong-Xin Liu, Caiping Tian, Baoyuan Qu, Chulei Gao, Peiyong Xin, Shujing Cheng, Wenjing Zhang, Pei Miao, Lei Li, Xiaojuan Zhang, Jinfang Chu,Jianru Zuo, Jiayang Li, Yang Bai, Xiaoguang Lei,* and Jian-Min Zhou*
Cell Host & Microbe 2020, 27(4), 601–613.e7
Plants deploy a variety of secondary metabolites to fend off pathogen attack. Although defense compounds are generally considered toxic to microbes, the exact mechanisms are often unknown. Here, we show that the Arabidopsis defense compound sulforaphane (SFN) functions primarily by inhibiting Pseudomonas syringae type III secretion system (TTSS) genes, which are essential for pathogenesis. Plants lacking the aliphatic glucosinolate pathway, which do not accumulate SFN, were unable to attenuate TTSS gene expression and exhibited increased susceptibility to P. syringae strains that cannot detoxify SFN. Chemoproteomics analyses showed that SFN covalently modified the cysteine at position 209 of HrpS, a key transcription factor controlling TTSS gene expression. Site-directed mutagenesis and functional analyses further confirmed that Cys209 was responsible for bacterial sensitivity to SFN in vitro and sensitivity to plant defenses conferred by the aliphatic glucosinolate pathway. Collectively, these results illustrate a previously unknown mechanism by which plants disarma pathogenic bacterium
Computation-Guided Development of the “Click” ortho-Quinone Methide Cycloaddition with Improved Kinetics
Xiaoyun Zhang, Shuo-Qing Zhang, Qiang Li, Fan Xiao, Zongwei Yue, Xin Hong,* and Xiaoguang Lei*
Org. Lett. 2020, 22, 8, 2920-2924
We report here a deep mechanistic study of the “click” ortho-quinone methide (oQM) cycloaddition between orthoquinolinone quinone methide (oQQM) and thio-vinyl ether (TV), named as TQ-ligation. DFT calculations revealed the unexpected fact that dehydration of oQQM precursors is the rate-determining step of this transformation, and two highly reactive oQQM
precursors were predicted. Guided by the calculations, a new “click” oQM cycloaddition which shows significantly improved kinetics and remarkable efficiency on protein labeling was developed.