Supramolecular Capsule Catalysis Enables the Exploration of Terpenoid Chemical Space Untapped by Nature.

Terpenes represent the largest and the most diverse class of natural compounds. This is remarkable as the whole variety is accessed from just a handful of highly conserved linear precursors. Modification of the cyclization precursors would enable a dramatic expansion of the accessible chemical space. However, natural enzymes do not enable us to tap into this potential, as they do not tolerate larger deviations from the prototypical substrate structure. Herein we report that supramolecular capsule catalysis enables facile access to diverse and novel terpenoid skeletons that formally can be traced back to C3-phenyl, benzyl, and homoprenyl derivatives of farnesol. Novel skeletons related to the presilphiperfolane core structure, as well as novel neoclovene derivatives were accessed efficiently in only four synthetic steps. Importantly, the products obtained carry functional groups that may be readily derivatized further.

Cytochrome P450 Mediated Cyclization in Eunicellane Derived Diterpenoid Biosynthesis.

Terpene cyclization, one of the most complex chemical reactions in nature, is generally catalyzed by two classes of terpene cyclases (TCs). Cytochrome P450s that act as unexpected TC-like enzymes are known but are very rare. In this study, we genome-mined a cryptic bacterial terpenoid gene cluster, named ari, from the thermophilic actinomycete strain Amycolatopsis arida. By employing a heterologous production system, we isolated and characterized three highly oxidized eunicellane derived diterpenoids, aridacins A-C (1-3), that possess a 6/7/5-fused tricyclic scaffold. In vivo and in vitro experiments systematically established a noncanonical two-step biosynthetic pathway for diterpene skeleton formation. First, a class I TC (AriE) cyclizes geranylgeranyl diphosphate (GGPP) into a 6/10-fused bicyclic cis-eunicellane skeleton. Next, a cytochrome P450 (AriF) catalyzes cyclization of the eunicellane skeleton into the 6/7/5-fused tricyclic scaffold through C2-C6 bond formation. Based on the results of quantum chemical computations, hydrogen abstraction followed by electron transfer coupled to barrierless carbocation ring closure is shown to be a viable mechanism for AriF-mediated cyclization. The biosynthetic logic of skeleton construction in the aridacins is unprecedented, expanding the catalytic capacity and diversity of P450s and setting the stage to investigate the inherent principles of carbocation generation by P450s in the biosynthesis of terpenoids.

Enantioselective Tail-to-Head Terpene Cyclizations by Optically Active Hexameric Resorcin[4]arene Capsule Derivatives.

Molecular capsules enable the conversion of substrates inside a closed cavity, mimicking to some extent enzymatic catalysis. Chirality transfer from the molecular capsule onto the encapsulated substrate has been only studied in a few cases. Here we demonstrate that chirality transfer is possible inside a rather large molecular container of approximately 1400 Å3 . Specifically, we present 1) the first examples of optically active hexameric resorcin[4]arene capsules, 2) their ability to enantioselectively catalyze tail-to-head terpene cyclizations, and 3) the surprisingly high sensitivity of enantioselectivity on the structural modifications.

Catalysis with Supramolecular Carbon-Bonding Interactions.

Herein, we describe a new catalysis platform, supramolecular carbon-bonding catalysis, which exploits the highly directional weak interactions between carbon centers of catalysts and electron donors to drive chemical reactions. To demonstrate this catalysis approach, we discovered a class of cyclopropane derivatives incorporated with carbonyl, ester and cyano groups as catalysts which showed general catalysis capability in different types of benchmark reactions. Among these typical examples, a challenging tail-to-head terpene cyclization can be achieved by supramolecular carbon-bonding catalysis. The co-crystal structures of catalyst and electron donors, comparison experiments, and titrations support a catalysis mode of carbon-bonding activation of Lewis basic reactants.

Molecular basis of triterpene-based chemophenetics in ferns.

MAIN CONCLUSION: A hypothesis that squalene cyclase genes are widely distributed throughout ferns was proposed. We successfully isolated a squalene cyclase pseudogene from a fern from which no triterpene hydrocarbons were detected Ferns are the most primitive vascular plants, with their locations ranging from tropical to cold temperate regions and from lowland to alpine zones. The triterpene hydrocarbons and their derivatives are characteristic fern metabolites, and are also chemophenetic markers. Recently, our biosynthetic study into fern squalene cyclases (SCs), the enzymes responsible for triterpene synthesis, gave an unexpected inconsistency between genotype (enzyme function) and chemotype (triterpene profile). This finding prompted us to propose a hypothesis that SC genes are widely distributed throughout ferns and lycophytes whether or not they produce triterpene hydrocarbons. To test this hypothesis, we employed a multifaceted approach based on phytochemical, biochemical, and phylogenetic analyses. As anticipated, we successfully isolated two SC pseudogenes from a fern from in which no or only one triterpene hydrocarbon was detected. Subsequent mutagenesis experiments resulted in the functional conversion of these pseudogenes into active SC genes. Given an auxiliary hypothesis regarding the inherent limit of the degenerate polymerase chain reaction (PCR) method, the overall dataset supported our hypothesis, although correction was required with respect to plant coverage. Not only did the corrected hypothesis outline the distribution of SC genes throughout ferns, it provided insight into the molecular basis of the triterpene-based chemophenetics in ferns, which is also discussed.

Sesquiterpene Cyclizations inside the Hexameric Resorcinarene Capsule: Total Synthesis of δ-Selinene and Mechanistic Studies.

The synthesis of terpene natural products remains a challenging task due to the enormous structural diversity in this class of compounds. Synthetic catalysts are unable to reproduce the tail-to-head terpene cyclization of cyclase enzymes, which create this diversity from just a few simple linear terpene substrates. Recently, supramolecular structures have emerged as promising enzyme mimetics. In the present study, the hexameric resorcinarene capsule was utilized as an artificial cyclase to catalyze the cyclization of sesquiterpenes. With the cyclization reaction as the key step, the first total synthesis of the sesquiterpene natural product δ-selinene was achieved. This represents the first total synthesis of a sesquiterpene natural product that is based on the cyclization of a linear terpene precursor inside a supramolecular catalyst. To elucidate the reaction mechanism, detailed kinetic studies and kinetic isotope measurements were performed. Surprisingly, the obtained kinetic data indicated that a rate-limiting encapsulation step is operational in the cyclization of sesquiterpenes.

Crystal structure and enantioselectivity of terpene cyclization in SAM-dependent methyltransferase TleD.

TleD is a SAM (S-adenosyl-l-methionine)-dependent methyltransferase and acts as one of the key enzymes in the teleocidin B biosynthesis pathway. Besides methyl transferring, TleD also rearranges the geranyl and indole moieties of the precursor to form a six-membered ring. Moreover, it does not show homologies with any known terpenoid cyclases. In order to elucidate how such a remarkable reaction could be achieved, we determined the complex crystal structures of TleD and the cofactor analogue S-adenosyl-l-homocysteine with or without the substrate teleocidin A1. A domain-swapped pattern via an additional N-terminal α-helix is observed in TleD hexamers. Structural comparison and alignment shows that this additional N-terminal α-helix is the common feature of SAM methyltransferase-like cyclases TleD and SpnF. The residue Tyr21 anchors the additional N-terminal α-helix to a 'core SAM-MT fold' and is a key residue for catalytic activity. Molecular dynamics simulation results suggest that the dihedral angle C23-C24-C25-C26 of teleocidin A1 is preferred to 60-90° in the TleD and substrate complex structure, which tend to adopt a Re-face stereocenter at C25 position after reaction and is according to in vitro enzyme reaction experiments. Our results also demonstrate that methyl transfer can be a new chemical strategy for carbocation formation in the terpene cyclization, which is the key initial step.

Onocerin Biosynthesis Requires Two Highly Dedicated Triterpene Cyclases in a Fern Lycopodium clavatum.

Onocerin is known for its unusual structure among triterpenoids, with a symmetrical structure that is formed by cyclizations at the both termini of dioxidosqualene. The nature of the enzyme catalyzing these unusual cyclizations has remained elusive for decades. Here, we report the cloning of genes responsible for these reactions; they exhibited unprecedented substrate specificities among oxidosqualene cyclase family members. Two genes, LCC and LCD, were identified from the fern Lycopodium clavatum. Expression in yeast revealed that both were required to produce α-onocerin. LCC, the first dioxidosqualene cyclase, catalyzed the production of a novel intermediate pre-α-onocerin from only dioxidosqualene as a substrate; LCD catalyzed the second half of the cyclization, exclusively from pre-α-onocerin. These results demonstrated that these two most unusual oxidosqualene cyclases were involved in onocerin biosynthesis.

Exploring the catalytic cascade of cembranoid biosynthesis by combination of genetic engineering and molecular simulations.

While chemical steps involved in bioactive cembranoid biosynthesis have been examined, the corresponding enzymatic mechanisms leading to their formation remain elusive. In the tobacco plant, Nicotiana tabacum, a putative cembratriene-ol synthase (CBTS) initiates the catalytic cascade that lead to the biosynthesis of cembratriene-4,6-diols, which displays antibacterial- and anti-proliferative activities. We report here on structural homology models, functional studies, and mechanistic explorations of this enzyme using a combination of biosynthetic and computational methods. This approach guided us to develop an efficient de novo production of five bioactive non- and monohydroxylated cembranoids. Our homology models in combination with quantum and classical simulations suggested putative principles of the CBTS catalytic cycle, and provided a possible rationale for the formation of premature olefinic side products. Moreover, the functional reconstruction of a N. tabacum-derived class II P450 with a cognate CPR, obtained by transcriptome mining provided for production of bioactive cembratriene-4,6-diols. Our combined findings provide mechanistic insights into cembranoid biosynthesis, and a basis for the sustainable industrial production of highly valuable bioactive cembranoids.