Direct Formation of Amide-Linked C-Glycosyl Amino Acids and Peptides via Photoredox/Nickel Dual Catalysis.

Glycoproteins account for numerous biological processes including those associated with diseases and infections. The advancement of glycopeptides has emerged as a promising strategy for unraveling biological pathways and discovering novel medicines. In this arena, a key challenge arises from the absence of efficient synthetic strategies to access glycopeptides and glycoproteins. Here, we present a highly concise approach to bridging saccharides with amino acids and peptides through an amide linkage. Our amide-linked C-glycosyl amino acids and peptides are synthesized through cooperative Ni-catalyzed and photoredox processes. The catalytic process generates a glycosyl radical and an amide carbonyl radical, which subsequently combine to yield the C-glycosyl products. The saccharide reaction partners encompass mono-, di-, and trisaccharides. All 20 natural amino acids, peptides, and their derivatives can efficiently undergo glycosylations with yields ranging from acceptable to high, demonstrating excellent stereoselectivities. As a substantial expansion of applications, we have shown that simple C-glycosyl amino acids can function as versatile building units for constructing C-glycopeptides with intricate spatial complexities.

Photocatalytic Direct para-selective C-H Amination of Benzyl Alcohols: Selectivity Independent of Side Substituents.

Aminoarenes are important molecules for broad applications in nearly all modern industries that involve chemicals. Direct and site-selective C-H bond amination of arenes provides the most efficient and convenient method to prepare aminoarenes. A main challenge is to selectively install the amino group (or other functional groups) to the distal para-carbon of arenes (especially multi-substituted arenes) during the C-H bond functionalization events. Herein, we address this problem by designing a new strategy via a sequential radical dearomatization/radical amination/rearomatization process for para-selective amination of benzyl alcohols. The para-selectivity of our reaction is completely independent of the electronic and steric properties of the other substituents of the arene substrates. Aminoarenes with many substituents (up to full substitution) and diverse substitution patterns, including those difficult to synthesize previously, could be readily prepared using our protocols. Further exploration of the current strategy shall lead to other challenging C-H functionalization of arenes.

Carbene-Catalyzed Atroposelective Annulation for Quick Access to Axially Chiral Thiazine Derivatives.

An N-heterocyclic carbene (NHC)-catalyzed atroposelective annulation reaction is disclosed for quick and efficient access to thiazine derivatives. A series of axially chiral thiazine derivatives bearing various substituents and substitution patterns were produced in moderate to high yields with moderate to excellent optical purities. Preliminary studies revealed that some of our products exhibit promising antibacterial activities against Xanthomonas oryzae pv. oryzae (Xoo) that causes rice bacterial blight.

Carbene-Catalyzed Activation of Remote Nitrogen Atoms of (Benz)imidazole-Derived Aldimines for Enantioselective Synthesis of Heterocycles.

A new mode of carbene-catalyzed heteroatom activation and asymmetric reactions is disclosed. The reaction starts with addition of a carbene catalyst to a (benz)imidazole-derived aldimine substrate. Subsequent oxidation and proton transfer lead to the formation of a catalyst-bound triaza-diene as the key intermediate, in which the nitrogen atom at a site remote to the catalyst-substrate bond is activated. This unusual triaza-diene intermediate then undergoes highly enantioselective reactions with activated ketones through a concerted asynchronous pathway, as supported by mechanistic studies and preliminary density function theory calculation.

Mining of UDP-glucosyltrfansferases in licorice for controllable glycosylation of pentacyclic triterpenoids.

Pentacyclic triterpenoids have wide applications in the pharmaceutical industry. The precise glucosylation at C-3 OH of pentacyclic triterpenoids mediated by uridine 5'-diphospho-glucosyltransferase (UDP-glucosyltransferase [UGT]) is an important way to produce valuable derivatives with various improved functions. However, most reported UGTs suffer from low regiospecificity toward the OH and COOH groups of pentacyclic triterpenoids, which significantly decreases the reaction efficiency. Here, two new UGTs (UGT73C33 and UGT73F24) were discovered in Glycyrrhiza uralensis. UGT73C33 showed high activity but poor regioselectivity toward the C-3 OH and C-30 COOH of pentacyclic triterpenoid, producing three glucosides. UGT73F24 showed rigid regioselectivity toward C-3 OH of typical pentacyclic triterpenoids producing only C-3 O-glucosylated derivatives. In addition, UGT73C33 and UGT73F24 showed a broad substrate scope toward typical flavonoids with various sugar donors. Next, the substrate recognition mechanism of UGT73F24 toward glycyrrhetinic acid (GA) and UDP-glucose was investigated. Two key residues, I23 and L84, were identified to determine activity, and site-directed mutagenesis of UGT73F24-I23G/L84N increased the activity by 4.1-fold. Furthermore, three in vitro GA glycosylation systems with UDP-recycling were constructed, and high yields of GA-3-O-Glc (1.25 mM), GA-30-O-Glc (0.61 mM), and GA-di-Glc (0.26 mM) were obtained. The de novo biosynthesis of GA-3-O-glucose (26.31 mg/L) was also obtained in engineered yeast.

CLE25 peptide regulates phloem initiation in Arabidopsis through a CLERK-CLV2 receptor complex.

The phloem, located within the vascular system, is critical for delivery of nutrients and signaling molecules throughout the plant body. Although the morphological process and several factors regulating phloem differentiation have been reported, the molecular mechanism underlying its initiation remains largely unknown. Here, we report that the small peptide-coding gene, CLAVATA 3 (CLV3)/EMBEYO SURROUNDING REGION 25 (CLE25), the expression of which begins in provascular initial cells of 64-cell-staged embryos, and continues in sieve element-procambium stem cells and phloem lineage cells, during post-embryonic root development, facilitates phloem initiation in Arabidopsis. Knockout of CLE25 led to delayed protophloem formation, and in situ expression of an antagonistic CLE25G6T peptide compromised the fate-determining periclinal division of the sieve element precursor cell and the continuity of the phloem in roots. In stems of CLE25G6T plants the phloem formation was also compromised, and procambial cells were over-accumulated. Genetic and biochemical analyses indicated that a complex, consisting of the CLE-RESISTANT RECEPTOR KINASE (CLERK) leucine-rich repeat (LRR) receptor kinase and the CLV2 LRR receptor-like protein, is involved in perceiving the CLE25 peptide. Similar to CLE25, CLERK was also expressed during early embryogenesis. Taken together, our findings suggest that CLE25 regulates phloem initiation in Arabidopsis through a CLERK-CLV2 receptor complex.

Cytological and Transcriptomic Analyses Reveal Important Roles of CLE19 in Pollen Exine Formation.

The CLAVATA3/ESR-RELATED (CLE) peptide signals are required for cell-cell communication in several plant growth and developmental processes. However, little is known regarding the possible functions of the CLEs in the anther. Here, we show that a T-DNA insertional mutant, and dominant-negative (DN) and overexpression (OX) transgenic plants of the CLE19 gene, exhibited significantly reduced anther size and pollen grain number and abnormal pollen wall formation in Arabidopsis (Arabidopsis thaliana). Interestingly, the DN-CLE19 pollen grains showed a more extensively covered surface, but CLE19-OX pollen exine exhibited clearly missing connections in the network and lacked separation between areas that normally form the lacunae. With a combination of cell biological, genetic, and transcriptomic analyses on cle19, DN-CLE19, and CLE19-OX plants, we demonstrated that CLE19-OX plants produced highly vacuolated and swollen aborted microspores (ams)-like tapetal cells, lacked lipidic tapetosomes and elaioplasts, and had abnormal pollen primexine without obvious accumulation of sporopollenin precursors. Moreover, CLE19 is important for the normal expression of more than 1,000 genes, including the transcription factor gene AMS, 280 AMS-downstream genes, and other genes involved in pollen coat and pollen exine formation, lipid metabolism, pollen germination, and hormone metabolism. In addition, the DN-CLE19(+/+) ams(-/-) plants exhibited the ams anther phenotype and ams(+/-) partially suppressed the DN-CLE19 transgene-induced pollen exine defects. These findings demonstrate that the proper amount of CLE19 signal is essential for the normal expression of AMS and its downstream gene networks in the regulation of anther development and pollen exine formation.

Radical Borylation/Cyclization Cascade of 1,6-Enynes for the Synthesis of Boron-Handled Hetero- and Carbocycles.

A synthetic method to construct boron-handled cyclic molecules was developed based on a radical borylation/cyclization cascade of 1,6-enynes. The process was initiated by the chemo- and regio-controlled addition of an N-heterocyclic carbene-boryl radical to an alkene or alkyne, followed by ring closure to afford boron-substituted cyclic skeletons. Further molecular transformations of the cyclic products to synthetically useful building blocks were also demonstrated.

EXO70A1-mediated vesicle trafficking is critical for tracheary element development in Arabidopsis.

Exocysts are highly conserved octameric complexes that play an essential role in the tethering of Golgi-derived vesicles to target membranes in eukaryotic organisms. Genes encoding the EXO70 subunit are highly duplicated in plants. Based on expression analyses, we proposed previously that individual EXO70 members may provide the exocyst with functional specificity to regulate cell type- or cargo-specific exocytosis, although direct evidence is not available. Here, we show that, as a gene expressed primarily during tracheary element (TE) development, EXO70A1 regulates vesicle trafficking in TE differentiation in Arabidopsis thaliana. Mutations of EXO70A1 led to aberrant xylem development, producing dwarfed and nearly sterile plants with very low fertility, reduced cell expansion, and decreased water potential and hydraulic transport. Grafting of a mutant shoot onto wild-type rootstock rescued most of these aboveground phenotypes, while grafting of a wild-type shoot to the mutant rootstock did not rescue the short root hair phenotype, consistent with the role of TEs in hydraulic transport from roots to shoots. Histological analyses revealed an altered pattern of secondary cell wall thickening and accumulation of large membrane-bound compartments specifically in developing TEs of the mutant. We thus propose that EXO70A1 functions in vesicle trafficking in TEs to regulate patterned secondary cell wall thickening.

CLE19 expressed in the embryo regulates both cotyledon establishment and endosperm development in Arabidopsis.

Embryo and endosperm development are two well co-ordinated developmental processes in seed formation; however, signals involved in embryo and endosperm interactions remain poorly understood. It has been shown before that CLAVATA3/ESR-RELATED 19 (CLE19) peptide is able to trigger root meristem consumption in a CLV2-dependent manner. In this study, the role of CLE19 in Arabidopsis seed development was explored using antagonistic peptide technology. CLE19 is expressed in the epidermal layers of the cotyledon primordia, hypocotyl, and root cap in the embryo. Transgenic plants carrying an antagonistic CLE19 G6T construct expressed under the control of CLE19 regulatory elements exhibited a dominant seed abortion phenotype, with defective cotyledon establishment in embryos and delayed nuclear proliferation and cellularization in endosperms. Ectopic expression of CLE19 G6T in Arabidopsis under the control of an endosperm-specific ALE1 promoter led to a similar defect in cotyledon establishment in embryos but without an evident effect on endosperm development. We therefore propose that CLE19 may act as a mobile peptide co-ordinating embryo and endosperm development.

Iron- or silver-catalyzed oxidative fluorination of cyclopropanols for the synthesis of ß-fluoroketones.

The Fe(III)- or Ag(I)-catalyzed oxidative fluorination of cyclopropanols via radical rearrangement is disclosed. This process features a straightforward and highly effective protocol for the site-specific synthesis of ß-fluoroketones and represents an expedient method for the synthesis of γ-, δ- and ε-fluoroketones. Notably, this reaction proceeds at room temperature and tolerates a diverse array of cyclopropanols.

Antagonistic peptide technology for functional dissection of CLV3/ESR genes in Arabidopsis.

In recent years, peptide hormones have been recognized as important signal molecules in plants. Genetic characterization of such peptides is challenging since they are usually encoded by small genes. As a proof of concept, we used the well-characterized stem cell-restricting CLAVATA3 (CLV3) to develop an antagonistic peptide technology by transformations of wild-type Arabidopsis (Arabidopsis thaliana) with constructs carrying the full-length CLV3 with every residue in the peptide-coding region replaced, one at a time, by alanine. Analyses of transgenic plants allowed us to identify one line exhibiting a dominant-negative clv3-like phenotype, with enlarged shoot apical meristems and increased numbers of floral organs. We then performed second dimensional amino acid substitutions to replace the glycine residue individually with the other 18 possible proteinaceous amino acids. Examination of transgenic plants showed that a glycine-to-threonine substitution gave the strongest antagonistic effect in the wild type, in which over 70% of transgenic lines showed the clv3-like phenotype. Among these substitutions, a negative correlation was observed between the antagonistic effects in the wild type and the complementation efficiencies in clv3. We also demonstrated that such an antagonistic peptide technology is applicable to other CLV3/EMBRYO SURROUNDING REGION (CLE) genes, CLE8 and CLE22, as well as in vitro treatments. We believe this technology provides a powerful tool for functional dissection of widely occurring CLE genes in plants.

Design, Synthesis, and Herbicidal Activity of Substituted 3-(Pyridin-2-yl)Phenylamino Derivatives.

To discover protoporphyrinogen oxidase (PPO) inhibitors with robust herbicidal activity and crop safety, three types of substituted 3-(pyridin-2-yl)phenylamino derivatives bearing amide, urea, or thiourea as side chain were designed via structure splicing strategy. Postemergence herbicidal activity assessment of 33 newly prepared compounds revealed that many of our compounds such as 6a, 7b, and 8d exhibited superior herbicidal activities against broadleaf and monocotyledon weeds to commercial acifluorfen. In particular, compound 8d exhibited excellent herbicidal activities and high crop safety at a dosage range of 37.5-150 g ai/ha. PPO inhibitory studies supported our compounds as typical PPO inhibitors. Molecular docking studies revealed that compound 8d provided effective interactions with Nicotiana tabacum PPO (NtPPO) via diverse interaction models, such as π-π stacking and hydrogen bonds. Molecular dynamics (MD) simulation studies and degradation studies were also conducted to gain insight into the inhibitory mechanism. Our study indicates that compound 8d may be a candidate molecule for the development of novel herbicides.

Discovery of Pyridyl-Benzothiazol Hybrids as Novel Protoporphyrinogen Oxidase Inhibitors via Scaffold Hopping.

In order to discover novel protoporphyrinogen oxidase (PPO) inhibitors with excellent herbicidal activity, a series of structurally novel 6-(pyridin-2-yl) benzothiazole derivatives were designed based on the scaffold hopping strategy. The in vitro experiments demonstrated that the newly synthesized compounds exhibited noteworthy inhibitory activity against Arabidopsis thaliana PPO (AtPPO), with IC50 values ranging from 0.06 to 1.36 µM. Preliminary postemergence herbicidal activity tests and crop safety studies indicated that some of our compounds exhibited excellent herbicidal activity and crop safety. For instance, compound (rac)-7as exhibited superior herbicidal activities to commercially available flumioxazin (FLU) and saflufenacil (SAF) at all the tested concentrations and showed effective herbicidal activities even at a dosage as low as 18.75 g ai/ha. Meanwhile, compound (rac)-7as showed good crop safety for wheat at a dosage as high as 150 g of ai/ha. Although the absolute configuration of compound 7as has no obvious effect on its herbicidal activity, compound (R)-7as showed a slightly higher crop safety than compound (S)-7as. Molecular simulation studies of Nicotiana tabacum PPO (NtPPO) and our candidate compounds showed that the benzothiazole moiety of compounds (R)-7as or (S)-7as formed multiple π-π stacking interactions with FAD, and the pyridine ring generated π-π stacking with Phe-392. Our finding proved that the pyridyl-benzothiazol hybrids are promising scaffolds for the development of PPO-inhibiting herbicides.

The sequence flanking the N-terminus of the CLV3 peptide is critical for its cleavage and activity in stem cell regulation in Arabidopsis.

BACKGROUND: Although it is known that CLAVATA3 (CLV3) acts as 12- and/or 13-amino acid (AA) secreted peptides to regulate the number of stem cells in shoot apical meristems (SAMs), how functional CLV3 peptides are generated and if any particular sequences are required for the processing remain largely unknown. RESULTS: We developed a mass spectrometry (MS)-based in vitro assay to monitor the cleavage of heterologously produced CLV3 fusion protein. Through co-cultivation of the fusion protein with Arabidopsis seedlings, we identified two cleavage sites: the previously reported one before Arg70 and a new one before Met39. Using synthetic peptides together with MALDI-Tof-MS analyses, we demonstrated that the non-conserved 5-AA motifs flanking N-termini of the CLV3 and its orthologous CLE1 peptides were critical for their cleavages and optimal activities in vitro. We also found that substitutions of Leu69 by Ala in fusion protein and in synthetic peptide of CLV3 compromised their cleavages, leading to significantly reduced activities in regulating the sizes of shoot and root meristems. CONCLUSIONS: These results suggest that 5-AA residues flanking the N-terminus of CLV3 peptide are required for proper cleavages and optimal function in stem cell regulation.

Construction of a UDP-Arabinose Regeneration System for Efficient Arabinosylation of Pentacyclic Triterpenoids.

Glycosylation is an important method of modifying natural products and is usually catalyzed by uridine 5'-diphosphate (UDP)-glycosyltransferase. UDP-ß-l-arabinose (UDP-Ara) confers specific functions to natural products such as pentacyclic triterpenoids. However, UDP-arabinosyltransferase with high regioselectivity toward pentacyclic triterpenoids has rarely been reported. In addition, UDP-Ara is mainly biosynthesized from UDP-α-d-glucose (UDP-Glc) through several reaction steps, resulting in the high cost of UDP-Ara. Herein, UGT99D1 was systematically characterized for specifically transferring one moiety of arabinose to the C-3 position of typical pentacyclic triterpenoids. Subsequently, 15 enzymes from plants, mammals, and microorganisms were characterized, and a four-enzyme cascade comprising sucrose synthase, UDP-Glc dehydrogenase, UDP-α-d-glucuronic acid decarboxylase, and UDP-Glc 4-epimerase was constructed to transform sucrose into UDP-Ara with UDP recycling. This system was demonstrated to efficiently produce the arabinosylated derivative (Ara-BA) of typical pentacyclic triterpenoid betulinic acid (BA). Finally, the in vitro cytotoxicity tests indicated that Ara-BA showed much higher anticancer activities than BA. The established arabinosylation platform shows the potential to enhance the pharmacological activity of natural products.

NHC-catalyzed [12+2] reaction of polycyclic arylaldehydes for access to indole derivatives.

An N-heterocyclic carbene (NHC) catalyzed enantio- and diastereoselective [12+2] cycloaddition is disclosed to rapidly construct sophisticated molecules bearing a tricyclic core and morpholine moiety. The success of our reaction relies on the NHC-catalyzed remote sp3 (C-H) bond activation of a 5H-benzo[a]pyrrolizine-3-carbaldehyde under oxidative conditions. Preliminary studies revealed that our products exhibit superior in vitro bioactivities against two plant pathogens to commercial Bismerthiazol (BT) and Thiodiazole Copper (TC).

Carbene and photocatalyst-catalyzed decarboxylative radical coupling of carboxylic acids and acyl imidazoles to form ketones.

The carbene and photocatalyst co-catalyzed radical coupling of acyl electrophile and a radical precursor is emerging as attractive method for ketone synthesis. However, previous reports mainly limited to prefunctionalized radical precursors and two-component coupling. Herein, an N-heterocyclic carbene and photocatalyst catalyzed decarboxylative radical coupling of carboxylic acids and acyl imidazoles is disclosed, in which the carboxylic acids are directly used as radical precursors. The acyl imidazoles could also be generated in situ by reaction of a carboxylic acid with CDI thus furnishing a formally decarboxylative coupling of two carboxylic acids. In addition, the reaction is successfully extended to three-component coupling by using alkene as a third coupling partner via a radical relay process. The mild conditions, operational simplicity, and use of carboxylic acids as the reacting partners make our method a powerful strategy for construction of complex ketones from readily available starting materials, and late-stage modification of natural products and medicines.

Direct Reaction of Nitroarenes and Thiols via Photodriven Oxygen Atom Transfer for Access to Sulfonamides.

Sulfonamide is a common motif in medicines and agrochemicals. Typically, this class of functional groups is prepared by reacting amines with sulfonyl chlorides that are presynthesized from nitro compounds and thiols, respectively. Here, we report a novel strategy that directly couples nitro compounds and thiols to form sulfonamides atom- and redox-economically. Mechanistic studies suggest our reaction proceeds via direct photoexcitation of nitroarenes that eventually transfers the oxygen atoms from the nitro group to the thiol unit.

Carbene-catalyzed atroposelective synthesis of axially chiral styrenes.

Axially chiral styrenes bearing a chiral axis between a sterically non-congested acyclic alkene and an aryl ring are difficult to prepare due to low rotational barrier of the axis. Disclosed here is an N-heterocyclic carbene (NHC) catalytic asymmetric solution to this problem. Our reaction involves ynals, sulfinic acids, and phenols as the substrates with an NHC as the catalyst. Key steps involve selective 1,4-addition of sulfinic anion to acetylenic acylazolium intermediate and sequential E-selective protonation to set up the chiral axis. Our reaction affords axially chiral styrenes bearing a chiral axis as the product with up to > 99:1 e.r., > 20:1 E/Z selectivity, and excellent yields. The sulfone and carboxylic ester moieties in our styrene products are common moieties in bioactive molecules and asymmetric catalysis.