Brewing coral terpenes-A yeast based approach to soft coral terpene cyclases.

Coral terpenes are important molecules with numerous applications. Here, we describe a robust and simple method to produce coral terpene scaffolds at scale. As an example of the approach, here we discover, express, and characterize further klysimplexin R synthases, expanding the known enzymology of soft coral terpene cyclases. We hope that the underlying method described will enable widespread basic research into the functions of coral terpenes and their biosynthetic genes, as well as the commercial development of biomedically and technologically important molecules.

Enantioselective Synthesis of (+)-Providencin and Its Unexpected Regioisomer via a Biomimetic Norrish-Yang Cyclization from (-)-Bipinnatin E.

A biomimetic semisynthesis of the diterpenoid (+)-providencin (2) and the unexpected novel C14 regioisomer 3 was achieved by photoirradiation of the proposed biosynthetic cembranoid precursor (-)-bipinnatin E (1). The absolute configuration assignments of 1 and 2 by correlation were established by X-ray analysis. A combination of NOESY data and photochemical reaction results revealed that both C2 and C14 positions of the macrocycle (-)-1 are suitable for hydrogen abstraction, thus affording an explanation to the mixture of cyclobutane photoproduct isomers obtained by a Norrish-Yang cyclization. These results also support the proposed biosynthetic hypothesis describing the genuine photochemical transformation of (-)-1 into (+)-2, without refuting that both regioisomer products 2/3 might be artifacts of isolation.

Biomimetic Approach to Diverse Coral Diterpenes from a Biosynthetic Scaffold.

Thousands of coral terpenes originate from simple scaffolds that undergo oxidative tailoring. While corals are excellent sources of drug leads, the challenge of supplying structurally complex drug leads from marine organisms has sometimes slowed their development. Making this even more challenging, in comparison to other organisms, such as plants and microbes, for which the terpene literature is substantial, very little is known about how the unique coral terpenes are biosynthesized and elaborated in nature. In this study, we used a semisynthetic strategy to produce at gram scale in yeast the eunicellane scaffold that underlies >200 coral compounds. Synthetic oxidation reactions were explored, generating key scaffolds that reflect three of the four structural classes derived from eunicellane and enabling the first asymmetric syntheses of the natural products solenopodin C and klysimplexin Q. Biomimetic methods and detailed mechanistic studies of synthetic reactions shed light on potential enzymological reactivity, including the role of epoxide rearrangement in eunicellane biosynthesis.

AgeMTPT, a Catalyst for Peptide N-Terminal Modification.

A key goal of synthetic biology is to enable designed modification of peptides and proteins, both in vivo and in vitro. N- and C-Terminal modification enzymes are crucial in this regard, but there are a few enzymatic options to protect peptide termini. AgeMTPT protects the N-terminus of short peptides with isoprene and the C-terminus as a methyl ester, but its substrate scope is unknown, limiting its application. Here, we investigate the substrate selectivity of the prenyltransferase domain, revealing a requirement for N-terminal aromatic amino acids, but with tolerance for diverse uncharged amino acids in the remaining positions. To demonstrate the potential of the enzyme, substrate selectivity data were used in the enzymatic modification of leu-enkephalin at the critical N-terminal residue. AgeMTPT active site mutagenesis led to an enzyme with expanded substrate scope, including the reverse geranylation of the N-termini of peptides. These data reveal potential applications of enzymatic peptide protection in synthetic biology.

Ancient defensive terpene biosynthetic gene clusters in the soft corals.

Diterpenes are major defensive small molecules that enable soft corals to survive without a tough exterior skeleton, and, until now, their biosynthetic origin has remained intractable. Furthermore, biomedical application of these molecules has been hampered by lack of supply. Here, we identify and characterize coral-encoded terpene cyclase genes that produce the eunicellane precursor of eleutherobin and cembrene, representative precursors for the >2,500 terpenes found in octocorals. Related genes are found in all sequenced octocorals and form their own clade, indicating a potential ancient origin concomitant with the split between the hard and soft corals. Eleutherobin biosynthetic genes are colocalized in a single chromosomal region. This demonstrates that, like plants and microbes, animals also harbor defensive biosynthetic gene clusters, supporting a recombinational model to explain why specialized or defensive metabolites are adjacently encoded in the genome.

Role of Macrocyclic Conformational Steering in a Kinetic Route toward Bielschowskysin.

Macrocyclic furanobutenolide-derived cembranoids (FBCs) are the biosynthetic precursors to a wide variety of highly congested and oxygenated polycyclic (nor)diterpenes (e.g. plumarellide, verrillin, and bielschowskysin). These architecturally complex metabolites are thought to originate from site-selective oxidation of the macrocycle backbone and a series of intricate transannular reactions. Yet the development of a common biomimetic route has been hampered by a lack of synthetic methods for the pivotal furan dearomatization in a regio- and stereoselective manner. To address these shortcomings, a concise strategy of epoxidation followed by a kinetically controlled furan dearomatization is reported. The surprising switch of facial α:ß-discrimination observed in the epoxidation of the most strained E-acerosolide versus E-deoxypukalide and E-bipinnatin J derived macrocycles has been rationalized by the variation of the 3D conformational landscape between macrocyclic scaffolds. A careful conformational analysis of these macrocycles by VT-NMR and NOESY experiments at low temperature was supported by DFT calculations to characterize these equilibrating macrocyclic conformers. The shift in conformational topology associated with a swing of the butenolide ring in E-deoxypukalide is in general agreement with the reversal of ß-selectivity observed in the epoxidation. We also describe the downstream functionalization of FBC-macrocycles and how the C-7 epoxide configuration is retentively translated to the C-3 stereogenicity in dearomatized products under kinetic control to secure the requisite 3S,7S,8S configurations for the bielschowskysin synthesis. Unlike previously speculated, our results suggest that the most strained FBC-macrocycles bearing a E-(Δ7,8)-alkene moiety may stand as the true biosynthetic precursors to bielschowskysin and several other polycyclic natural products of this class.

A Kinetic Dearomatization Strategy for an Expedient Biomimetic Route to the Bielschowskysin Skeleton.

Bielschowskysin (1), the flagship of the furanocembranoid diterpene family, has attracted attention from chemists owing to its intriguing and daunting polycyclic architecture and medicinal potential against lung cancer. The high level of functionalization of 1 poses a considerable challenge to synthesis. Herein, a stereoselective furan dearomatization strategy of furanocembranoids was achieved via the intermediacy of chlorohydrins. The stereochemical course of the kinetic dearomatization was established, and the C3 configuration of the resulting exo-enol ether intermediates proved to be essential to complete the late-stage transannular [2+2] photocycloaddition. Overall, this biomimetic strategy starting from the natural product acerosolide (9) featured an unprecedented regio- and highly stereoselective furan dearomatization, which provided rapid access to the pivotal exo-enol ethers en route to the intricate bielschowskyane skeleton.

Cyclized 9,11-secosterol enol-ethers from the gorgonian Pseudopterogorgia americana.

Chemical investigation of the MeOH extract from the gorgonian Pseudopterogorgia americana afforded two rare sterols, ameristerenol A (1) and B (2), both 9,11-secosterols possesses a seven-membered cyclic enol-ether in ring C, and ameristerol A (3) is the first example of a naturally occurring 9,11-secosterol containing a gorgosterol side chain with a C-24(28) double bond. Ameristerenol A (1) was converted to the sterol derivatives 4-6 to provide additional chemical diversity and comparison for biological screening. The structures of compounds 1-6, along with three related known analogues 7-9, were determined on the basis of extensive spectroscopic analysis and by comparison with literature data. Compound 6 exhibited slight cytotoxicity activity against human breast cancer cell line MDA-MB-231.