Enzymatic biosynthesis of novel 2-(2-phenylethyl)chromone glycosides catalyzed by UDP-glycosyltransferase UGT71BD1.

2-(2-Phenylethyl)chromones (PECs) are the main bioactive components of agarwood which showed diverse pharmaceutical activities. Glycosylation is a useful structural modification method to improve compounds' druggability. However, PEC glycosides were rarely reported in nature which largely limited their further medicinal investigations and applications. In this study, the enzymatic glycosylation of four naturally separated PECs 1-4 was achieved using a promiscuous glycosyltransferase UGT71BD1 identified from Cistanche tubulosa. It could accept UDP-Glucose, UDP-N-acetylglucosamine and UDP-xylose as sugar donors and conduct the corresponding O-glycosylation of 1-4 with high conversion efficiencies. Three O-glucosylated products 1a (5-hydroxy-2-(2-phenylethyl)chromone 8-O-ß-D-glucopyranoside), 2a (8-chloro-2-(2-phenylethyl)chromone 6-O-ß-D-glucopyranoside) and 3a (2-(2-phenylethyl)chromone 6-O-ß-D-glucopyranoside) were prepared and structurally elucidated as novel PEC glucosides based on NMR spectroscopic analyses. Subsequent pharmaceutical evaluation found that 1a showed remarkably improved cytotoxicity against HL-60 cells, whose cell inhibition rate was 19 times higher than that of its aglycon 1. The IC50 value of 1a was further determined to be 13.96 ± 1.10 µM, implying its potential as a promising antitumor-leading candidate. To improve the production of 1, docking, simulation and site-directed mutagenesis were performed. The important role of P15 in the glucosylation of PECs was discovered. Besides, a mutant K288A with a two-fold increased yield for 1a production was also afforded. This research reported the enzymatic glycosylation of PECs for the first time, and also provide an eco-friendly pathway for the alternative production of PEC glycosides for leading compounds discovery.

[Research progress on application of multi-enzyme-catalyzed cascade reactions in enzymatic synthesis of natural products].

As a biocatalyst, enzyme has the advantages of high catalytic efficiency, strong reaction selectivity, specific target products, mild reaction conditions, and environmental friendliness, and serves as an important tool for the synthesis of complex organic molecules. With the continuous development of gene sequencing technology, molecular biology, genetic manipulation, and other technologies, the diversity of enzymes increases steadily and the reactions that can be catalyzed are also gradually diversified. In the process of enzyme-catalyzed synthesis, the majority of common enzymatic reactions can be achieved by single enzyme catalysis, while many complex reactions often require the participation of two or more enzymes. Therefore, the combination of multiple enzymes together to construct the multi-enzyme cascade reactions has become a research hotspot in the field of biochemistry. Nowadays, the biosynthetic pathways of more natural products with complex structures have been clarified, and secondary metabolic enzymes with novel catalytic activities have been identified, discovered, and combined in enzymatic synthesis of natural/unnatural molecules with diverse structures. This study summarized a series of examples of multi-enzyme-catalyzed cascades and highlighted the application of cascade catalysis methods in the synthesis of carbohydrates, nucleosides, flavonoids, terpenes, alkaloids, and chiral molecules. Furthermore, the existing problems and solutions of multi-enzyme-catalyzed cascade method were discussed, and the future development direction was prospected.

Glycosylation of Aromatic Glycosides by a Promiscuous Glycosyltransferase UGT71BD1 from Cistanche tubulosa.

Multiple-glycosylated glycosides are a major source of bioactive leads. However, most of the currently reported glycosyltransferases (GTases) mainly catalyze glycosylation of aglycones without sugar group substitution. GTases accepting diverse glycosides as substrates are rarely reported. In this article, a new GTase UGT71BD1 was identified from Cistanche tubulosa, a desert herb plant abundant with various phenylethanoid glycosides (PhGs). Interestingly, UGT71BD1 showed no activity toward the aglycone of PhGs. Instead, it could catalyze the further glycosylation of PhG compounds to produce new phenylethanoid multiglycosylated glycosides, including the natural rarely separated tetraglycoside PhGs. Extensive assays found the unprecedented substrate promiscuity of UGT71BD1 toward diverse glycosides including flavonoid glycosides, stilbene glycosides, and coumarin glycosides, performing further mono- or diglycosylation with efficient conversion rates. Using UGT71BD1, six multiglycosylated glycosides were prepared and structurally identified by NMR spectroscopy. These products showed enhanced pharmacological activities compared with the substrates. Docking, dynamic simulation, and mutagenesis studies identified key residues for UGT71BD1's activity and revealed that the sugar modules in glycosides play crucial roles in substrate recognition, thus partly illuminating the unusual substrate preference of UGT71BD1 toward diverse glycosides. UGT71BD1 could be a potential enzyme tool for glycosylation of diverse glycosides.

The RLA1/SMOS1 Transcription Factor Functions with OsBZR1 to Regulate Brassinosteroid Signaling and Rice Architecture.

Brassinosteroids (BRs) are plant-specific steroid hormones that control plant growth and development. Recent studies have identified key components of the BR signaling pathway in Arabidopsis thaliana and in rice (Oryza sativa); however, the mechanism of BR signaling in rice, especially downstream of GSK3/SHAGGY-like kinase (GSK2), remains unclear. Here, we identified a BR-insensitive rice mutant, reduced leaf angle1 (rla1), and cloned the corresponding gene. RLA1 was identical to the previously reported SMALL ORGAN SIZE1 (SMOS1), which was cloned from another allele. RLA1/SMOS1 encodes a transcription factor with an APETALA2 DNA binding domain. Genetic analysis indicated that RLA1/SMOS1 functions as a positive regulator in the BR signaling pathway and is required for the function of BRASSINAZOLE-RESISTANT1 (OsBZR1). In addition, RLA1/SMOS1 can interact with OsBZR1 to enhance its transcriptional activity. GSK2 can interact with and phosphorylate RLA1/SMOS1 to reduce its stability. These results demonstrate that RLA1/SMOS1 acts as an integrator of the transcriptional complex directly downstream of GSK2 and plays an essential role in BR signaling and plant development in rice.

Asymmetric synthesis of (-)-solanidine and (-)-tomatidenol.

A concise asymmetric synthesis of two naturally occurring seco-type cholestane alkaloids (-)-solanidine and (-)-tomatidenol from (-)-diosgenin with a linear reaction sequence of 12 steps and 13 steps, respectively, is reported. The synthetic strategy includes the highly controlled establishment of highly functionalized octahydroindolizine ((-)-solanidine) and 1-oxa-6-azaspiro[4.5]decane ((-)-tomatidenol) cores with five stereocenters, respectively, from (-)-diosgenin, featuring two stereoselective cascade transformations including a modified cascade ring-switching process of furostan-26-acid to open the E-ring of (-)-diosgenin and a cascade azide reduction/intramolecular reductive amination to close the E- and F-rings of (-)-solanidine and (-)-tomatidenol. This work should enable further explorations of chemical and biological spaces based on solanidine, tomatidenol and related natural products.

Correction: Asymmetric synthesis of (-)-solanidine and (-)-tomatidenol.

Correction for 'Asymmetric synthesis of (-)-solanidine and (-)-tomatidenol' by Yun Wang et al., Org. Biomol. Chem., 2020, 18, 3169-3176, DOI: .

Facile Access to Bridged Ring Systems via Point-to-Planar Chirality Transfer: Unified Synthesis of Ten Cyclocitrinols.

Bridged ring systems are found in a wide variety of biologically active molecules including pharmaceuticals and natural products. However, the development of practical methods to access such systems with precise control of the planar chirality presents considerable challenges to synthetic chemists. In the context of our work on the synthesis of cyclocitrinols, a family of steroidal natural products, we herein report the development of a point-to-planar chirality transfer strategy for preparing bridged ring systems from readily accessible fused ring systems. Inspired by the proposed pathway for biosynthesis of cyclocitrinols from ergosterol, our strategy involves a bioinspired cascade rearrangement, which enabled the gram-scale synthesis of a common intermediate in nine steps and subsequent unified synthesis of 10 cyclocitrinols in an additional one to three steps. Our work provides experimental support for the proposed biosynthetic pathway and for the possible interrelationships between members of the cyclocitrinol family. In addition to being a convenient route to 5(10→19) abeo-steroids, our strategy also offers a generalized approach to bridged ring systems via point-to-planar chirality transfer. Mechanistic investigations suggest that the key cascade rearrangement involves a regioselective ring scission of a cyclopropylcarbinyl cation rather than a direct Wagner-Meerwein rearrangement.

Scalable Synthesis of Cyclocitrinol.

A 10-step synthesis of the C25 steroid natural product cyclocitrinol from inexpensive, commercially available pregnenolone is reported. This synthesis features a biomimetic cascade rearrangement to efficiently construct the challenging bicyclo[4.4.1] A/B ring system, which enabled a gram-scale synthesis of the bicyclo[4.4.1] enone intermediate 18 in only nine steps. This work also provides experimental support for the biosynthetic origin of cyclocitrinol.

Alkynes From Furans: A General Fragmentation Method Applied to the Synthesis of the Proposed Structure of Aglatomin†B.

Furans are versatile synthons in organic chemistry. Described is a general method for transforming furans into alkynes by dual C-C double-bond cleavage. The reaction is proposed to proceed by sequential [4+2] cycloaddition between furan and singlet oxygen and a formal retro-(3+2) fragmentation of the endoperoxide intermediate. A wide array of furans, including those derived from sapogenins, are amenable to this reaction, thus providing the corresponding alkynoic acids in up to 88 % yields. The synthetic utility was demonstrated by a seven-step synthesis of the proposed structure of a pregnane natural product, aglatomin B, from a known intermediate.

Synthesis of 12,12'-azo-13,13'-diepi-Ritterazine N.

A synthesis of the 12,12'-azo-analogue of ritterazine N from hecogenin is reported. Ring contraction of two 6/5 bicyclic ring systems, one trans-fused and another spiro, to 5/5 spiro ring systems is accomplished with excellent stereochemical control. Key transformations include an abnormal Baeyer-Villiger oxidation, a Norrish type I cleavage, an intramolecular dipolar [3 + 2] cycloaddition, and an intramolecular oxymecuration. Failing to uncover the ß-OH ketone from the isoxazoline ring, we end up with a synthesis of a cyclic analogue of ritterazine N.

Constructing 24(23→22)-abeo-Cholestane from Tigogenin in a 20(22→23)-abeo-Way via a PhI(OAc)2-mediated Favorskii Rearrangement.

Transforming tigogenin, a steroidal sapogenin, to a 24(23→22)-abeo-cholestane, which is an unusual structural feature shared by the aglycons of saundersiosides and candicanoside A, is described. The spiroketal of tigogenin was unfolded and the resulting C22-ketone was subjected to Favorskii rearrangement mediated by PhI(OAc)2/KOH/MeOH to squeeze out the C22 from the side chain, thus reaching the 24(23→22)-abeo-cholestane structure.

Divergent Synthesis of Solanidine and 22-epi-Solanidine.

A divergent synthesis of solanidine and 22-epi-solanidine, two 25S natural steroidal alkaloids, from 25R-configured diosgenin acetate, is described. Initially, solanidine was synthesized through a series of transformations including a cascade ring-switching process of furostan-26-acid, an epimerization of C25 controlled by the conformation of six-membered lactone ring, an intramolecular Schmidt reaction, and an imine reduction/intramolecular aminolysis process. To address the epimerization issue during Schmidt reaction, an improved synthesis was developed, which also led to a synthesis of 22-epi-solanidine. In this synthesis, selective transformation of azido lactone to azido diol and amino diol was realized through a reduction relay tactic. The azido diol was transformed to solanidine via an intramolecular Schmidt reaction/N-alkylation/reduction process and to 22-epi-solanidine via an intramolecular double N-alkylation process.

Molecular characterization and structure basis of a malonyltransferase with both substrate promiscuity and catalytic regiospecificity from Cistanche tubulosa.

Enzymatic malonylation of natural glycosides provides a promising alternative method for drug-like malonylated glycosides supply. However, the catalytic potential and structural basis of plant malonyltransferase are far from being fully elucidated. This work identified a new malonyltransferase CtMaT1 from Cistanche tubulosa. It displayed unprecedented mono- and/or di-malonylation activity toward diverse glucosides with different aglycons. A "one-pot" system by CtMaT1 and a malonyl-CoA synthetase was established to biosynthesize nine new malonylated glucosides. Structural investigations revealed that CtMaT1 possesses an adequately spacious acyl-acceptor pocket capable of accommodating diverse glucosides. Additionally, it recognizes malonyl-CoA through strong electrotactic and hydrogen interactions. QM/MM calculation revealed the H167-mediated SN2 reaction mechanism of CtMaT1, while dynamic simulations detected the formation of stable hydrogen bonds between the glucose-6-OH group and H167, resulting in its high malonylation regiospecificity. Calculated energy profiles of two isomeric glycosides highlighted lower reaction energy barriers towards glucoside substrates, emphasizing CtMaT1's preference for glucosides. Furthermore, a mutant CtMaT1H36A with notably increased di-malonylation activity was obtained. The underlying molecular mechanism was illuminated through MM/GBSA binding free energy calculation. This study significantly advances the understanding of plant acyltransferases from both functional and protein structural perspectives, while also providing a versatile tool for enzymatic malonylation applications in pharmacology.

Biosynthesis Investigations of Terpenoid, Alkaloid, and Flavonoid Antimicrobial Agents Derived from Medicinal Plants.

The overuse of antibiotics in the past decades has led to the emergence of a large number of drug-resistant microorganisms. In recent years, the infection rate caused by multidrug-resistant microorganisms has been increasing, which has become one of the most challenging problems in modern medicine. Plant-derived secondary metabolites and their derivatives have been identified to display significant antimicrobial abilities with good tolerance and less adverse side effects, potentially having different action mechanisms with antibiotics of microbial origin. Thus, these phyto-antimicrobials have a good prospect in the treatment of multidrug-resistant microorganisms. Terpenoids, alkaloids, and flavonoids made up the predominant part of the currently reported phytochemicals with antimicrobial activities. Synthetic biology research around these compounds is one of the hotspot fields in recent years, which not only has illuminated the biosynthesis pathways of these phyto-antimicrobials but has also offered new methods for their production. In this review, we discuss the biosynthesis investigations of terpenoid, alkaloid, and flavonoid antimicrobial agents-using artemisinin and oleanolic acid (terpenoids), berberine and colchicine (alkaloids), and baicalin (flavonoids) as examples-around their antimicrobial action mechanisms, biosynthesis pathway elucidation, key enzyme identification, and heterologous production, in order to provide useful hints for plant-derived antimicrobial agent discovery and development.

Total synthesis of (±)-spiniferin-1 via a polyfluoroalkanosulfonyl fluoride induced homoallylic carbocation rearrangement reaction.

A facile total synthesis of marine natural product (±)-spiniferin-1 has been accomplished in eight steps with 28.9% overall yield, involving a rearrangement reaction initiated by polyfluoroalkanosulfonyl fluoride to construct the 1,6-methano[10]annulene core of the natural product as a key step.

Stereoselective Synthesis of (-)-Verazine and Congeners via a Cascade Ring-Switching Process of Furostan-26-acid.

An efficient synthetic strategy for three natural seco-type cholestane alkaloids isolated from the Veratrum plants, based on commercially available naturally occurring and abundant (-)-diosgenin (1), as exemplified in the concise asymmetric synthesis of (-)-verazine (4), (-)-veramiline (5) (proposed structure), and its 22-epimer, (-)-oblonginine (6), is presented. This work highlights the application of a cascade ring-switching process of (-)-diosgenin to achieve the E-ring opening and construction of chiral six-membered lactone challenges in seco-type cholestane alkaloid synthesis. This approach enables the synthesis of related natural and nature-like novel cholestane alkaloids, opening up opportunities for more extensive exploration of cholestane alkaloid biology.

Synthesis Toward and Stereochemical Assignment of Clathsterol: Exploring Diverse Strategies to Polyoxygenated Sterols.

Herein we describe a synthesis of the trisulfate derivative of clathsterol (1), a marine sterol endowed with impressive structural features and moderate inhibitory activity against HIV-1 reverse transcriptase. By synthesizing two possible isomers of the side chain, the stereochemistry of 1 is assigned. In creating chiral side chains from steroidal lactone, our strategies, including an addition/reduction procedure to give C22R-OH, an epoxide-opening reaction, and a [3.3]-rearrangement to induce the generation of C24S-Et and C24R-Et respectively, are highly flexible and complementary to each other.

Synthesis of the aglycon of aspafiliosides E and F based on cascade reactions.

A synthesis of C17α-OH-tigogenin, the aglycon of aspafiliosides E and F, is described. The main features of the synthesis are three cascade processes, which involve the iodo-lactonization of furostan-26-acid to open ring E, a cascade hydrolysis/intramolecular SN2 process to close ring E, and a cascade intramolecular redox-ketalization process to close ring F. This synthesis would enrich the strategies used for the manipulation of spiroketals in steroidal sapogenins and other substrates.

Synthesis of Demissidine and Solanidine.

Demissidine and solanidine, two steroidal alkaloids, are synthesized in eight steps from tigogenin acetate and diosgenin acetate, respectively, which involve the replacement of three C-O bonds with C-N bonds. Key transformations include a cascade ring-switching process of furostan-26-acid, an epimerization of C25, an intramolecular Schmidt reaction, and an imine reduction/intramolecular aminolysis process.