Chemo-enzymatic total synthesis: current approaches toward the integration of chemical and enzymatic transformations

• Ryo Tanifuji and
• Hiroki Oguri

Beilstein J. Org. Chem. 2024, 20, 1693–1712, doi:10.3762/bjoc.20.151

• considered a remarkable example of utilizing the complementarity between chemical and enzymatic transformations (Scheme 1 and Scheme 2). The cotylenin and fusicoccin families comprise structurally related diterpene glucosides with a 5/8/5 fused tricyclic aglycon and a sugar moiety linked through the C9

• hydroxy group of the aglycon. Cotylenin exhibits promising anticancer activity, and its diterpene aglycon, cotylenol (1), was isolated from the filamentous fungi such as Phomopsis amygdali and Cladosporium sp. 501-7W (Scheme 2A) [20][21][22]. Brassicicenes, differing in oxidation levels from 1, have been

Computation-guided scaffold exploration of 2E,6E-1,10-trans/cis-eunicellanes

• Zining Li,
• Sana Jindani,
• Volga Kojasoy,
• Teresa Ortega,
• Erin M. Marshall,
• Khalil A. Abboud,
• Sandra Loesgen,
• Dean J. Tantillo and
• Jeffrey D. Rudolf

Beilstein J. Org. Chem. 2024, 20, 1320–1326, doi:10.3762/bjoc.20.115

• natural products containing a foundational 6/10-bicyclic framework and can be divided into two main classes, cis and trans, based on the configurations of their ring fusion at C1 and C10. Previous studies on two bacterial diterpene synthases, Bnd4 and AlbS, revealed that these enzymes form cis- and trans

• rearrangement; DFT calculations; diterpene; electrophilic cyclization; eunicellane; Introduction The eunicellane diterpenoids are a family of nearly 400 natural products that present a conserved 6/10-bicyclic hydrocarbon framework [1][2][3]. Mostly known from soft corals [2], but with a growing number of

• ) of alkenes in the 10-membered ring (Figure 1A). During biosynthesis, the eunicellane skeleton is first constructed by terpene synthases that cyclize the diterpene precursor geranylgeranyl diphosphate (Figure 1B) [5][6][7][8][9]. The latter two structural elements, the configurations of the 6/10

Stability trends in carbocation intermediates stemming from germacrene A and hedycaryol

• Naziha Tarannam,
• Prashant Kumar Gupta,
• Shani Zev and
• Dan Thomas Major

Beilstein J. Org. Chem. 2024, 20, 1189–1197, doi:10.3762/bjoc.20.101

• monoterpenes (C10), sesquiterpenes (C15), and diterpenes (C20). Enzymes such as monoterpene, sesquiterpene, and diterpene synthases act on geranyl diphosphate (GPP), farnesyl diphosphate (FPP), and geranylgeranyl diphosphate (GGPP) to yield mono-, sesqui-, and diterpenes, respectively. These linear precursors

Enhancing structural diversity of terpenoids by multisubstrate terpene synthases

• Min Li and
• Hui Tao

Beilstein J. Org. Chem. 2024, 20, 959–972, doi:10.3762/bjoc.20.86

• enzymes (Table 1) [18]. A TPS-f subfamily enzyme CoTPS5 from Cananga odorata has been characterized to convert 3 to (E)-β-ocimene (9), 4 to 8, and 5 to diterpene α-springene (10) (Figure 2) [22]. Both in vitro assays and in vivo transgenic expression of CoTPS5 confirmed the absence of side products

• substrates, MSTSs can also accept partially cyclized substrates. A class I diterpene synthase SiTPS8 from Setaria italicais is capable of utilizing three copalyl pyrophosphate (CPP) stereoisomers that were generated by different class II TSs, including ent-CPP (19), (+)-CPP (20), and syn-CPP (21), to

• generate different diterpene skeletons 22–28, which were further converted to diterpenoids by a P450 monooxygenase (CYP99A17) (Table 1, Figure 2) [20]. These findings will enable further investigation of the functions of terpenoids in plants and crops. MSTSs from fungi Fungi are also prolific producers of

Confirmation of the stereochemistry of spiroviolene

• Yao Kong,
• Yuanning Liu,
• Kaibiao Wang,
• Tao Wang,
• Chen Wang,
• Ben Ai,
• Hongli Jia,
• Guohui Pan,
• Min Yin and
• Zhengren Xu

Beilstein J. Org. Chem. 2024, 20, 852–858, doi:10.3762/bjoc.20.77

• cyclization mechanism of spiroviolene. Keywords: boron migration; diterpene; spiroviolene; stereochemistry; Introduction Terpenes represent one of the most fascinating families of natural products due to their structural complexity and diversity, as well as their indispensable biological functions that

• for GGPP production. Also, we have cloned the SvS-coding gene directly from Streptomyces violens CGMCC 4.1786 (= NRRL ISP-5597) into pET28a to give pET28a-svs. The resultant two plasmids were then co-transformed into commercially available E. coli BL21(DE3) for diterpene production. Spiroviolene could

Genome mining of labdane-related diterpenoids: Discovery of the two-enzyme pathway leading to (−)-sandaracopimaradiene in the fungus Arthrinium sacchari

• Fumito Sato,
• Terutaka Sonohara,
• Shunta Fujiki,
• Akihiro Sugawara,
• Yohei Morishita,
• Taro Ozaki and
• Teigo Asai

Beilstein J. Org. Chem. 2024, 20, 714–720, doi:10.3762/bjoc.20.65

• synthesize a diterpene skeleton: After the initial cyclization of GGPP possibly catalyzed by a putative class II enzyme AsCPS, the resulting labdane or related skeleton would undergo cyclization by a putative class I enzyme AsPS. To assess this hypothesis, we conducted the functional characterization of AsPS

Recent developments in the engineered biosynthesis of fungal meroterpenoids

• Zhiyang Quan and
• Takayoshi Awakawa

Beilstein J. Org. Chem. 2024, 20, 578–588, doi:10.3762/bjoc.20.50

• , the combinatorial biosynthesis of diterpene pyrones, derived from a pyrone skeleton and a C20 terpenoid skeleton, has been achieved by combining multiple biosynthetic pathways in a fungal heterologous expression host [16]. First, subA (PKS), subC (PT), subD (GGPP synthase), subE (FMO), and subB (CYC

• combinatorial biosynthesis of diterpene pyrone meroterpenoids. The production of subglutinol A by SubABCDE + DpasF (A), the production of novel diterpene pyrones by SubABCDE + various tailoring enzymes (B), and the production of schearone A by SubACD + EsdpE + EsdpB (C). The biosynthetic reaction from the

Unraveling the role of prenyl side-chain interactions in stabilizing the secondary carbocation in the biosynthesis of variexenol B

• Moe Nakano,
• Rintaro Gemma and
• Hajime Sato

Beilstein J. Org. Chem. 2023, 19, 1503–1510, doi:10.3762/bjoc.19.107

• biosynthesis of trichobrasilenol [22], by combined methods of computational and experimental chemistry. Recently, Dickschat et al. reported the synthesis of a novel diterpene compound, variexenol B, using a substrate analogue called iso-GGPP (Scheme 1) [23]. This biosynthetic pathway has two interesting

Functional characterisation of twelve terpene synthases from actinobacteria

• Anuj K. Chhalodia,
• Houchao Xu,
• Georges B. Tabekoueng,
• Binbin Gu,
• Kizerbo A. Taizoumbe,
• Lukas Lauterbach and
• Jeroen S. Dickschat

Beilstein J. Org. Chem. 2023, 19, 1386–1398, doi:10.3762/bjoc.19.100

• phylogenetically related enzymes were in one case not expressed and in two cases inactive, suggesting pseudogenisation in the respective branch of the phylogenetic tree. Furthermore, a diterpene synthase for allokutznerene and a sesterterpene synthase for sesterviolene were identified. Keywords: actinomycetes

• from the same branch from S. subrutilus, S. natalensis and S. violens were in one case not expressed and in two cases only yielded soluble, but inactive enzymes with any of the tested substrates GPP, FPP, GGPP and GFPP (Table 1, entries 11–13). Diterpene and sesterterpene synthases One more terpene

• inactive enzymes were obtained and one enzyme was not expressed. Another interesting discovery was the identification of a diterpene synthase from Kutzneria kofuensis that selectively produces allokutznerene. This compound was previously only known as a side product from a closely related phomopsene

Cassane diterpenoids with α-glucosidase inhibitory activity from the fruits of Pterolobium macropterum

• Sarot Cheenpracha,
• Ratchanaporn Chokchaisiri,
• Lucksagoon Ganranoo,
• Sareeya Bureekaew,
• Thunwadee Limtharakul and
• Surat Laphookhieo

Beilstein J. Org. Chem. 2023, 19, 658–665, doi:10.3762/bjoc.19.47

• , 167.0, two olefinics at δC 155.4, 128.0, and three oxygenated at δC 104.1, 71.3, 65.4). Assuming a cassane-type diterpene skeleton, the 1H and 13C NMR spectra (Table 1) displayed two sets (A and B) of the characteristic signals of seven methyl singlets [set A: δH/δC 1.20 (s, H3-20)/16.7, 1.00 (s, H3-19

• acetoxy groups [set A: δH 2.06 (s, 6-OCOCH3)/δC 21.8, and 170.7; set B: δH 2.10 (s, 6′-OCOCH3)/δC 21.9 and 170.4]. Careful analysis of the NMR data indicated the presence of a dimeric cassane-type diterpene skeleton whose NMR spectra resembled those of pterolobirin B [11], an unprecedented caged cassane

Enolates ambushed – asymmetric tandem conjugate addition and subsequent enolate trapping with conventional and less traditional electrophiles

• Péter Kisszékelyi and
• Radovan Šebesta

Beilstein J. Org. Chem. 2023, 19, 593–634, doi:10.3762/bjoc.19.44

• war against resistant bacteria strains. The tricyclic diterpene fungal metabolite (+)-pleuromutilin was isolated in 1951 [104]. Since then it has served as a starting point for developing new antibiotics, including semisynthetic derivatives effective against Gram-positive or even both types of

• particularly difficult targets for synthetic chemists. Nevertheless, Huang and co-workers have successfully resolved the total synthesis of waihoensene which was isolated in 1997 from a New Zealand podocarp [110]. This diterpene has a unique substructure with fused 5-membered rings in an angular fashion and

• Peyssonnelia sp. This diterpene showed promising biological activity against methicillin-resistant Staphylococcus aureus (MRSA) and liver-stage Plasmodium berghei. Structurally, peyssonnoside A belongs to a new class of diterpene glycosides with a distinctive tetracyclic carbon skeleton. From the point of view

Strategies to access the [5-8] bicyclic core encountered in the sesquiterpene, diterpene and sesterterpene series

• Cécile Alleman,
• Charlène Gadais,
• Laurent Legentil and
• François-Hugues Porée

Beilstein J. Org. Chem. 2023, 19, 245–281, doi:10.3762/bjoc.19.23

• compound 24 was rationalized by an isomerization of the double bond prior to the cyclization step. Isolated from the Ecuadorian liverwort Anastrophyllum auritum in 1994, ent-fusicoauritone (28) is a diterpene presenting a fusicoccan skeleton with a β-hydroxyketone on the A-ring and 5 stereogenic centers

Combining the best of both worlds: radical-based divergent total synthesis

• Kyriaki Gennaiou,
• Antonios Kelesidis,
• Maria Kourgiantaki and
• Alexandros L. Zografos

Beilstein J. Org. Chem. 2023, 19, 1–26, doi:10.3762/bjoc.19.1

• to hypertensive properties [24][25][26] depending on the subtle substituents in the periphery of a decalin core (Scheme 2). In 2018, the Baran group reported the divergent total synthesis of several pyrone diterpene natural products, relying solely on one-electron-based retrosynthesis. The group

Total synthesis of grayanane natural products

• Nicolas Fay,
• Rémi Blieck,
• Cyrille Kouklovsky and
• Aurélien de la Torre

Beilstein J. Org. Chem. 2022, 18, 1707–1719, doi:10.3762/bjoc.18.181

• a synthetic pathway also giving access to several diterpene glycosides close to pierisformaside C in order to study the biological activity of this family. Their strategy was based on a common forward intermediate and a late construction of the central seven-membered ring. In the beginning of the

Characterization of a new fusicoccane-type diterpene synthase and an associated P450 enzyme

• Jia-Hua Huang,
• Jian-Ming Lv,
• Liang-Yan Xiao,
• Qian Xu,
• Fu-Long Lin,
• Gao-Qian Wang,
• Guo-Dong Chen,
• Sheng-Ying Qin,
• Dan Hu and
• Hao Gao

Beilstein J. Org. Chem. 2022, 18, 1396–1402, doi:10.3762/bjoc.18.144

• nature and possess a variety of biological activities. Up to date, only five fusicoccane-type diterpene synthases have been identified. Here, we identify a two-gene biosynthetic gene cluster containing a new fusicoccane-type diterpene synthase gene tadA and an associated cytochrome P450 gene tadB from

• Talaromyces wortmannii ATCC 26942. Heterologous expression reveals that TadA catalyzes the formation of a new fusicoccane-type diterpene talaro-7,13-diene. D2O isotope labeling combined with site-directed mutagenesis indicates that TadA might employ a different C2,6 cyclization strategy from the known

• fusicoccane-type diterpene synthases, in which a neutral intermediate is firstly formed and then protonated by an environmental proton. In addition, we demonstrate that the associated cytochrome P450 enzyme TadB is able to catalyze multiple oxidation of talaro-7,13-diene to yield talaro-6,13-dien-5,8-dione

Understanding the competing pathways leading to hydropyrene and isoelisabethatriene

• Shani Zev,
• Marion Ringel,
• Ronja Driller,
• Bernhard Loll,
• Thomas Brück and
• Dan T. Major

Beilstein J. Org. Chem. 2022, 18, 972–978, doi:10.3762/bjoc.18.97

• main GGPP cyclization products, along with two minor compounds, namely the isoelisabethatrienes (IEs) A (13%) and B (9%), respectively. Interestingly, the elisabethatriene diterpene macrocycle and its isoforms can act as biosynthetic precursors of the bioactive compounds erogorgiaene and pseudopterosin

Efficient production of clerodane and ent-kaurane diterpenes through truncated artificial pathways in Escherichia coli

• Fang-Ru Li,
• Xiaoxu Lin,
• Qian Yang,
• Ning-Hua Tan and
• Liao-Bin Dong

Beilstein J. Org. Chem. 2022, 18, 881–888, doi:10.3762/bjoc.18.89

• -kaurane skeletons outlined here may provide an attractive route to prepare other privileged diterpene scaffolds. Keywords: artificial pathway; ent-kaurene; Escherichia coli; overproduction; terpentetriene; Introduction Diterpenoids, of which there are over 34,000 members (http://terokit.qmclab.com

• ), have attracted great attention from chemists and biologists due to their intriguing chemical structures and broad pharmacological functions [1][2][3][4]. The vast structural diversity of diterpenoids arise biosynthetically from the following two stages: i) diterpene synthase (DTS, also called diterpene

• group or a combination of enzymatic and chemical tools (chemoenzymatic synthesis) [8][9][10][11]. Despite great efforts spanning several decades, de novo organic synthetic methods access to the core diterpene skeletons are still highly challenging owing to their numerous chiral centers and polycyclic

Terpenoids from Glechoma hederacea var. longituba and their biological activities

• Dong Hyun Kim,
• Song Lim Ham,
• Zahra Khan,
• Sun Yeou Kim,
• Sang Un Choi,
• Chung Sub Kim and
• Kang Ro Lee

Beilstein J. Org. Chem. 2022, 18, 555–566, doi:10.3762/bjoc.18.58

• . longituba (common name: ground ivy) has been used for the treatment of asthma, bronchitis, cholelithiasis, colds, and inflammation. In the present study, three new sesquiterpene glycosides (1–3), two new diterpene glycosides (4 and 5), and four known compounds (6–9) were isolated from its MeOH extract. A

• . Interestingly, of the two diterpene glucosides (4 and 5) that were structurally similar except for the C-2' functionality (4, -OH; 5, -OAc) of the glucopyranosyl group, only compound 5 showed NO inhibitory activity. This suggests that the presence of an acetyl group at C-2' might play an important role in the

• against SK-MEL-2 cell line, with an IC50 value of 9.81 μM. Conclusion Nine terpene derivatives, including three new sesquiterpene glycosides (1–3), two new diterpene glycosides (4 and 5), a known diterpene (6), and three known triterpenes (7–9) were isolated from CHCl3-, EtOAc-, and n-BuOH-soluble layers

Four bioactive new steroids from the soft coral Lobophytum pauciflorum collected in South China Sea

• Di Zhang,
• Zhe Wang,
• Xiao Han,
• Xiao-Lei Li,
• Zhong-Yu Lu,
• Bei-Bei Dou,
• Wen-Ze Zhang,
• Xu-Li Tang,
• Ping-Lin Li and
• Guo-Qiang Li

Beilstein J. Org. Chem. 2022, 18, 374–380, doi:10.3762/bjoc.18.42

• metabolites with great variety and bioactivities. Previous chemical studies on soft corals Lobophytum, widely distributed in the world, resulted in the identification of lobane diterpene [1], cembranoids [2], and biscembranoids [3] with different bioactivities. Moreover, structurally specific steroids

Targeting active site residues and structural anchoring positions in terpene synthases

• Anwei Hou and
• Jeroen S. Dickschat

Beilstein J. Org. Chem. 2021, 17, 2441–2449, doi:10.3762/bjoc.17.161

• , turning SmTS1 from a sesterterpene into a diterpene synthase. This article gives rational explanations for these findings that may generally allow for protein engineering of other terpene synthases to improve their catalytic efficiency or to change their functions. Keywords: biosynthesis; enzyme

• ). SmTS1 has a low amino acid sequence identity to other characterised TPSs, with the diterpene synthase (DTS) for cattleyene from Streptomyces cattleya as one of the closest relatives, which shows only 29% sequence identity [17]. We have recently shown that the sum of the calculated van der Waals volumina

• C3 of the substrate (G182 in Figure 1) and assists in substrate ionisation. The introduction of steric bulk at this position blocked this movement for SdS, resulting in inactivity [11]. Diterpene synthase activity of SmTS1 variants While the A222V enzyme variant did not convert GFPP, presumably

Volatile emission and biosynthesis in endophytic fungi colonizing black poplar leaves

• Christin Walther,
• Pamela Baumann,
• Katrin Luck,
• Beate Rothe,
• Peter H. W. Biedermann,
• Jonathan Gershenzon,
• Tobias G. Köllner and
• Sybille B. Unsicker

Beilstein J. Org. Chem. 2021, 17, 1698–1711, doi:10.3762/bjoc.17.118

• , and heterologously expressed in Escherichia coli. To determine mono-, sesqui-, and diterpene-forming activity, the bacterial raw protein extracts were assayed with the substrates GPP, FPP, and GGPP, each in the presence of the co-substrate magnesium chloride. Both protein extracts containing the

NMR Spectroscopy of supramolecular chemistry on protein surfaces

• Peter Bayer,
• Anja Matena and
• Christine Beuck

Beilstein J. Org. Chem. 2020, 16, 2505–2522, doi:10.3762/bjoc.16.203

• ligand consisting of a fusicoccane diterpene core combined with different sugar moieties was designed to improve its properties as molecular glue for the 14-3-3/p53 complex [105]. Upon peptide binding in the absence of ligand, the 14-3-3 resonances of the binding site show reduced signal intensities. The

Understanding the role of active site residues in CotB2 catalysis using a cluster model

• Keren Raz,
• Ronja Driller,
• Thomas Brück,
• Bernhard Loll and
• Dan T. Major

Beilstein J. Org. Chem. 2020, 16, 50–59, doi:10.3762/bjoc.16.7

• , Dept. of Chemistry, Technical University of Munich (TUM), Lichtenbergstr. 4, 85748 Garching, Germany 10.3762/bjoc.16.7 Abstract Terpene cyclases are responsible for the initial cyclization cascade in the multistep synthesis of a large number of terpenes. CotB2 is a diterpene cyclase from Streptomyces

• cascade can provide important information towards a biosynthetic strategy for cyclooctatin and the biomanufacturing of related terpene structures. Keywords: active site; CotB2 cyclase; diterpene; mechanism; quantum mechanics; Introduction Enzymes catalyze numerous complex biochemical reactions in

• crystal structure of a diterpene cyclase was reported by Christianson and co-workers [22]. These structures, in conjunction with extensive biochemical work [10][13][14][23], have contributed to the understanding of mechanistic details of terpene cyclases and facilitated rational enzyme design [24

Bacterial terpene biosynthesis: challenges and opportunities for pathway engineering

• Eric J. N. Helfrich,
• Geng-Min Lin,
• Christopher A. Voigt and
• Jon Clardy

Beilstein J. Org. Chem. 2019, 15, 2889–2906, doi:10.3762/bjoc.15.283

• inverted direction of charge propagation along the oligoisoprene chain in the cyclization cascade [53]. The resulting cyclized product retains the pyrophosphate moiety and can serve as the substrate for a second cyclization catalyzed by type I TCs to generate even more complicated diterpene backbones

• , simply by rationally mutating the active-site residues. The majority of bacterial type II diterpene TCs produce bicyclic labdane, halimadane, or clerodane skeletons with different stereochemistry, levels of unsaturation, and hydroxylation patterns [124], which undergo further conversion with their

• cognate type I diterpene TCs. It was therefore hypothesized that promiscuous type I TCs might be able to process different type II products to generate non-natural structural diversity. Indeed, pairing of type II TCs with type I TCs generated 13, 41, and 49 novel terpene skeletons, respectively, in three

Current understanding and biotechnological application of the bacterial diterpene synthase CotB2

• Ronja Driller,
• Daniel Garbe,
• Norbert Mehlmer,
• Monika Fuchs,
• Keren Raz,
• Dan Thomas Major,
• Thomas Brück and
• Bernhard Loll

Beilstein J. Org. Chem. 2019, 15, 2355–2368, doi:10.3762/bjoc.15.228

• date, CotB2 represents the best studied bacterial diterpene synthase. Its reaction mechanism has been addressed by isoptope labeling, targeted mutagenesis and theoretical computations in the gas phase, as well as full enzyme molecular dynamic simulations. By X-ray crystallography different snapshots of

• mutagenesis have exciting applications for the sustainable production of high value bioactive substances. Keywords: biotechnology; CotB2; crystal structure; cyclooctatin; diterpene; reaction mechanism; terpene synthase; Introduction Terpenes represent one of the most diverse groups of natural biomolecules

• will focus particularly on bacterial diterpene synthases, in context with other sesqui- and ditperpene synthases of bacterial, fungal and plant origin. The initial step in diterpene biosynthesis (Figure 1) is the incremental condensation of dimethylallyl diphosphate (1) and isopentylen diphosphate (2