Rapid Synthesis of Chiral Figure-Eight Macrocycles Using a Preorganized Natural Product-Based Scaffold.

Chiral D2 -symmetric figure-eight shaped macrocycles are promising scaffolds for amplifying the chiroptical properties of π-conjugated systems. By harnessing the inherent and adaptable conformational dynamics of a chiral C2 -symmetric bispyrrolidinoindoline (BPI) manifold, we developed an enantio-divergent modular synthetic platform to rapidly generate a diverse range of chiral macrocycles, spanning from 14- to 66-membered rings, eliminating the need for optical resolution. Notably, a 32-membered figure-eight macrocycle showed excellent circularly polarized luminescence (CPL: |glum |=1.1×10-2 ) complemented by a robust emission quantum yield (Φfl =0.74), to achieve outstanding CPL brightness (BCPL : ϵ×Φfl ×|glum |/2=480). Using quadruple Sonogashira couplings, this versatile synthetic platform enables precise adjustments of the angle, distance, and length among intersecting π-conjugated chromophores. Our synthetic strategy offers a streamlined and systematic approach to significantly enhance BCPL values for a variety of chiral D2 -symmetric figure-eight macrocycles.

Synthetic Modulation of an Unstable Dehydrosecodine-type Intermediate and Its Encapsulation into a Confined Cavity Enable Its X-ray Crystallographic Observation.

Numerous indole alkaloids such as the iboga- and aspidosperma-type are believed to be biosynthesized via a common hypothetical intermediate, dehydrosecodine. The highly reactive nature of dehydrosecodine-type compounds has hampered their isolation and structural elucidation. In this study, we achieved the first X-ray structural determination of a dehydrosecodine-type compound by integrating synthetic optimization of the reactivity and stabilizing the fragile molecule by encapsulation into a supramolecular host. Formation of a 1 : 1 complex of the dehydrosecodine-type labile guest bearing both vinyl indole and dihydropyridine units with the host was observed. This integrated approach not only provides insights into the biosynthetic conversions but also allows stabilization and storage of the reactive and otherwise short-lived intermediate within the confined hydrophobic cavity.

Synthesis of a Halicyclamine-Type Macrocyclic Scaffold via Biomimetic Transannular Cyclization.

Previous attempts for biomimetic transannular reactions between two dihydropyrdine (DHP) rings incorporated in macrocycle frameworks relevant to manzamine alkaloids have been unsuccessful. Herein, we report an efficient regiocontrolled transannular cyclization of C2-symmetric macrocyclic precursor with rational modifications of the 3 and 6 positions of the DHP rings to synthesize a halicyclamine-type scaffold. The cis-double bonds installed in the macrocyclic alkyl loops played a crucial role in achieving the biomimetic transannular cyclization.

Regiocontrolled Dimerization of Densely Functionalized 1,6-Dihydropyridines for the Biomimetic Synthesis of a Halicyclamine-type Scaffold by Preventing Disproportionation.

The biomimetic dimerization of 1,6-dihydropyridines (DHPs) remains a daunting challenge due to competitive disproportionation pathways. Herein we report the regioselective dimerization of densely functionalized 1,6-DHPs that allow direct access to the bis-nitrogen bicyclic scaffold of halicyclamines. Disproportionation triggered by the hydride shift of 1,6-DHP was suppressed by the use of geminal disubstituted substrates. Installation of an electron-withdrawing substituent at the C3 position was demonstrated to be crucial for facilitating biomimetic dimerization under metal-free conditions, with exquisite control of regioselectivity at ambient temperature. Our approach, featuring an appropriately functionalized and substantially stabilized substrate rather than merely adopting the highly reactive and labile hypothetical biosynthetic intermediate, allowed gram-scale and atom-economical synthesis of the bis-nitrogen bicyclic scaffold. Furthermore, conversion of a series of 1,6-DHPs provided mechanistic insights by circumventing the competitive disproportionation reaction. This revealed not only the innate reactivity of the conjugate diene system for [4 + 2] cycloaddition but also the reversibility of the dimerization reaction with multiple cationic intermediates in equilibrium.

Design and synthesis of 14 and 15-membered macrocyclic scaffolds exhibiting inhibitory activities of hypoxia-inducible factor 1α.

Inspired by the privileged molecular skeletons of 14- and 15-membered antibiotics, we adopted a relatively unexplored synthetic approach that exploits alkaloidal macrocyclic scaffolds to generate modulators of protein-protein interactions (PPIs). As mimetics of hot-spot residues in the α-helices responsible for the transcriptional regulation, three hydrophobic sidechains were displayed on each of the four distinct macrocyclic scaffolds generating diversity of their spatial arrangements. Modular assembly of the building blocks followed by ring-closing olefin metathesis reaction and subsequent hydrogenation allowed concise and divergent synthesis of scaffolds 1-4. The 14-membered alkaloidal macrocycles 2-4 demonstrated similar inhibition of hypoxia-inducible factor (HIF)-1α transcriptional activities (IC50 between 8.7 and 10 µM), and 4 demonstrated the most potent inhibition of cell proliferation in vitro (IC50 = 12 µM against HTC116 colon cancer cell line). A docking model suggested that 4 could mimic the LLxxL motif in HIF-1α, in which the three sidechains are capable of matching the spatial arrangements of the protein hot-spot residues. Unlike most of the stapled peptides, the 14-membered alkaloidal scaffold has a similar size to the α-helix backbone and does not require additional atoms to induce α-helix mimetic structure. These experimental results underscore the potential of alkaloidal macrocyclic scaffolds featuring flexibly customizable skeletal, stereochemical, substitutional, and conformational properties for the development of non-peptidyl PPI modulators targeting α-helix-forming consensus sequences responsible for the transcriptional regulation.

A fully synthetic 6-aza-artemisinin bearing an amphiphilic chain generates aggregates and exhibits anti-cancer activities.

Installation of a nitrogen at the C6 position of artemisinin facilitates the addition of a functional unit on the cyclohexane moiety (C-ring). In this study, conjugation of an amphiphilic chain, composed of sequentially connected hydrophilic oligoethylene glycol, hydrophobic alkyl chain, urea, and 4,4'-disubstituted biphenyl linker, imparted self-assembling properties. The fully synthetic mid-molecular weight 6-aza-artemisinin 6 bearing the amphiphilic moiety formed aggregates (approx. 200 nm) at ambient temperature and exhibited increased in vitro anti-cancer activities compared to the N-benzylated aza-artemisinin 5.

Rapid and Systematic Exploration of Chemical Space Relevant to Artemisinins: Anti-malarial Activities of Skeletally Diversified Tetracyclic Peroxides and 6-Aza-artemisinins.

To achieve both structural changes and rapid synthesis of the tetracyclic scaffold relevant to artemisinins, we explored two kinds of de novo synthetic approaches that generate both skeletally diversified tetracyclic peroxides and 6-aza-artemisinins. The anti-malarial activities of the tetracyclic peroxides with distinct skeletal arrays, however, were moderate and far inferior to artemisinins. Given the privileged scaffold of artemisinins, we next envisioned element implantation at the C6 position with a nitrogen without the trimmings of substituents and functional groups. This molecular design allowed the deep-seated structural modification of the hitherto unexplored cyclohexane moiety (C-ring) while keeping the three-dimensional structure of artemisinins. Notably, this approach induced dramatic changes of retrosynthetic transforms that allow an expeditious catalytic asymmetric synthesis with generation of substitutional variations at three sites (N6, C9, and C3) of the 6-aza-artemisinins. These de novo synthetic approaches led to the lead discovery with substantial intensification of the in vivo activities, which undermine the prevailing notion that the C-ring of artemisinins appears to be merely a structural unit but to be a functional area as the anti-malarial pharmacophore. Furthermore, we unexpectedly found that racemic 6-aza-artemisinin (33) exerted exceedingly potent in vivo efficacies superior to the chiral one and the first-line drug, artesunate.

Total synthesis of alkaloids using both chemical and biochemical methods.

Covering: 2000 to 2019Rapid access to genomic data has facilitated the identification of the biosynthetic enzyme genes of alkaloid natural products and elucidation of their biosynthetic pathways. Enzymes for the rapid construction of molecular scaffolds and versatile modifications during the late-stage biosynthesis of complex molecular skeletons constitute unique features of biosynthetic machineries. For example, enzymes involved in an alkaloid biosynthesis. In this review, we discuss three types of useful enzymes and enzymatic reactions that have been found in the biosynthetic studies of several alkaloids, and discuss their applications for the total synthesis of natural alkaloids and their derivatives. The selected examples include a single non-ribosomal peptide synthetase SfmC that catalyzes key Pictet-Spengler reactions, which construct a characteristic tetrahydroisoquinoline skeleton in antitumor antibiotics such as saframycin, prenylation-oxidative modification enzymes involved in the biosynthesis of fungal tremorgenic mycotoxins such as penitrem as well as versatile Diels-Alderases recently discovered in the biosynthesis of plant monoterpene indole alkaloids of iboga and aspidosperma type.

Zn(OTf)2-mediated annulations of N-propargylated tetrahydrocarbolines: divergent synthesis of four distinct alkaloidal scaffolds.

Intramolecular hydroarylations of N-propargylated tetrahydrocarbolines were efficiently mediated using a unique combination of Zn(OTf)2 with t-BuOH under neutral conditions. Use of the artificial force induced reaction method in the global reaction route mapping strategy provided insights into the Zn(OTf)2-mediated hydroarylations and the associated intriguing solvent effects of t-BuOH facilitating a protodezincation process without a Brønsted acid activator. We systematically implemented three distinct hydroarylations as well as an unanticipated α-alkenylation of a carbonyl group to obtain the four alkaloidal scaffolds 2-4, and 18. Zn(OTf)2-mediated annulation of 1c proceeded through kinetic formation of the spiroindole 3c followed by an alkenyl shift and concomitant retro-Mannich-type fragmentation to furnish azepino[4,5-b]indole 2 framework. Substituents on substrate 1 in the vicinity of the reaction sites substantially affected the mode of the divergent annulations. Judicious choices of the substituents, solvent and reaction conditions enabled programmable divergent synthesis of the four distinct skeletons.

Generation of C5-desoxy analogs of tetrahydroisoquinoline alkaloids exhibiting potent DNA alkylating ability.

C5-desoxy analogs of tetrahydroisoquinoline (THIQ) alkaloids were designed and synthesized as hitherto unexplored structural variants for evaluation of their DNA alkylating activities. While chemical synthesis of the C5-desoxy analogs bearing a phenolic hydroxyl group in the A-ring of the saframycins was assumed to be laborious based on semi-synthetic modifications, a chemo-enzymatic approach allowed for concise access to the analogs. The C5-desoxy analog 7 exhibited greater DNA alkylating ability with a wider tolerance for the sequence variations compared to cyanosafracin B. The C5-desoxy A-ring having a C8 phenolic hydroxyl group, and a C1 substituent in the vicinity of the C21 aminonitrile responsible for DNA alkylation, were demonstrated to play pivotal roles in the interaction between the THIQ alkaloids and DNA.

Stereodivergent Synthesis of Bispyrrolidinoindoline Alkaloidal Scaffolds and Generation of a Lead Candidate with Stereospecific Antiproliferative Activity.

The fungal secondary metabolites (+)-WIN 64821 and (-)-ditryptophenaline are biosynthesized through condensation of l-tryptophan and l-phenylalanine, followed by reductive dimerization with generation of stereochemical variations. Inspired by the stereodivergent biogenetic process, we designed and synthesized a collection of bispyrrolidinoindoline diketopiperazine alkaloids and their analogues with systematic diversification of the stereochemistry of the privileged structural motif of the fungal alkaloids. Not only the stereochemical modifications of (+)-WIN 64821 at the 3-/3'-, 11-/11'-, and 15-/15'-positions, but also ring cleavage of the diketopiperazine moieties, allowed the generation of a lead compound exhibiting potent growth inhibitory activity (IC50 =3.03 µm) toward human colon cancer cells. Structure-activity relationship studies revealed that all six stereogenic centers were essential for the pharmacophore. High cell densities dramatically intensified the cytotoxic activities of the lead compound.

Chiroptical Inversion of Europium(III) Complexes by Changing a Remote Stereogenic Center of a C2-Symmetric Bispyrrolidinoindoline Manifold.

As an effort to integrate natural products chemistry and coordination chemistry, a diastereomeric pair of chiral alkaloidal manifolds composed of a bispyrrolidinoindoline (BPI) framework was designed and synthesized to generate luminescent EuIII complexes with switchable chiroptical properties. The C2-symmetric alkaloidal manifolds were linked with bis(benzimidazolyl)pyridine (BBIPy) as an achiral metal-binding component through substituents installed at the stereogenic 2/2' positions of the BPI manifolds. The resulting diastereomeric pair of ligands, syn-L1 and anti-L2, allow pseudomirror symmetrical presentation of the metal-binding BBIPy units due to the stereogenic centers on the alkaloidal manifold. The ligand syn-L1 induces intramolecular coordination to form the 1:1 complex EuIII( syn-L1) composed of a single stranded metal helicate which exhibits a negative split Cotton effect. In contrast, the ligand anti-L2 led to a supramolecular assembly comprising the 2:2 complex EuIII2( anti-L2)2 consisting of a bimetallic double-stranded helicate which shows a positive split Cotton effect. Thus, the sp3 stereogenic centers in the BPI manifolds play pivotal roles in controlling both metal-ligand equilibria and chirality-switching of luminescent EuIII complexes. This approach, which exploits diastereomeric natural product-based manifolds, provides a relatively unexplored means for diversifying metal coordination modes and for controlling the chiroptical properties of the resultant luminescent lanthanoid complexes.

Design of C2-symmetric alkaloidal chiral amphiphiles and configurational effects on self-assembly.

Alkaloids are a cornerstone in the development of medicinal and synthetic compounds due to their capability of specific recognition of targeted biomacromolecules, and uses in optical resolution and asymmetric reactions. To explore the untapped potential of the rigid and densely functionalized structures of alkaloids with precisely regulated configurations as optically active core scaffolds of self-assembling molecules, here we report the design, syntheses, chiroptical properties and self-assemblies of C2-symmetric alkaloidal amphiphiles with anti/syn stereochemical variations. Bispyrrolidinoindoline (BPI) was chosen as the optically active core scaffold. It was synthetically modified with hydrophobic alkyl chains and hydrophilic tetraethylene glycol tails to provide amphiphilicity. The anti/syn configurational differences in the amphiphiles significantly influenced the chiroptical, dynamic and supramolecular properties. Amphiphiles with anti-configurations responded to a solvent polarity change by altering their conformations, while the conformational changes of the syn-type amphiphiles were largely restricted. Furthermore, the anti-type amphiphile having the highest structural flexibility showed a characteristic split Cotton effect in an organic medium and formed the largest aggregates upon addition of water with a significant change in the circular dichroism (CD) profile, while amphiphiles having conformational restriction by the syn-configuration or a macrocyclic structure showed monomodal CD signals and afforded significantly smaller aggregates upon addition of water. Hence, the C2-symmetric alkaloidal BPI structure is demonstrated to be a useful core scaffold for supramolecular chemistry to design amphiphiles with controllable configurational diversity, which allows for the customization of chiroptical properties, conformational flexibility and self-assembly.

Chemo-enzymatic Total Syntheses of Jorunnamycin A, Saframycin A, and N-Fmoc Saframycin Y3.

The antitumor tetrahydroisoquinoline (THIQ) alkaloids share a common pentacyclic scaffold that is biosynthesized by nonribosomal peptide synthetases involving unique enzymatic Pictet-Spengler cyclizations. Herein we report concise and divergent chemo-enzymatic total syntheses of THIQ alkaloids by merging precise chemical synthesis with in vitro engineered biosynthesis. A recombinant enzyme SfmC responsible for the biosynthesis of saframycin A was adapted for the assembly of these natural products and their derivatives, by optimizing designer substrates compatible with SfmC through chemical synthesis. The appropriately functionalized pentacyclic skeleton were efficiently synthesized by streamlining the linkage between SfmC-catalyzed multistep enzymatic conversions and chemical manipulations of the intermediates to install aminonitrile and N-methyl groups. This approach allowed rapid access to the elaborated pentacyclic skeleton in a single day starting from two simple synthetic substrates without isolation of the intermediates. Further functional group manipulations allowed operationally simple and expeditious syntheses of jorunnamycin A, saframycin A, and N-Fmoc saframycin Y3 that could be versatile and common precursors for the artificial production of other antitumor THIQ alkaloids and their variants.

Design and De Novo Synthesis of 6-Aza-artemisinins.

Development of designer natural product variants, 6-aza-artemisinins, enabled us to achieve structural modification of the hitherto unexplored cyclohexane moiety of artemisinin and concise de novo synthesis of the tetracyclic scaffold in just four steps from the modular assembly of three simple building blocks. This expeditious catalytic asymmetric synthetic approach generated lead candidates exhibiting superior in vivo antimalarial activities to artemisinin.

Stereo-controlled synthesis of functionalized tetrahydropyridines based on the cyanomethylation of 1,6-dihydropyridines and generation of anti-hepatitis C virus agents.

Densely functionalized tetrahydropyridines were stereoselectively synthesized from 1,6-dihydropyridines. Exploiting a carbonyl group installed at the C3 position of the 1,6-dihydropyridine system, we devised a strategy for cyanomethylation at C2/C6 and subsequent divergent installation of an allyl group at C3/C5 in a highly regio- and stereo-controlled manner. This versatile protocol for programmable functionalization of the 1,6-dihydropyridine system allows the divergent and streamlined synthesis of multiply-substituted tetrahydropyridines as an important class of biologically and medicinally relevant scaffolds. Two of the N-heterocyclic compounds bearing an alkyl nitrile group showed anti-hepatitis C virus (HCV) activity.

Biomimetic Assembly Lines Producing Natural Product Analogs: Strategies from a Versatile Manifold to Skeletally Diverse Scaffolds.

Biosynthetic assembly lines have evolved in nature, adopting divergent processes to produce a vast number of secondary metabolites. Inspired by these biogenetic processes, this account introduces recent investigations by my research group to formulate a synthetic strategy for establishing a biomimetic assembly line. With the aim not only to construct natural product-relevant scaffolds within 5-7 steps, but also to systematically diversify skeletal and stereochemical properties and functional groups, divergent synthetic processes exploiting a versatile manifold have been developed. This approach allows for cost-effective production of skeletally diverse and biologically active natural product analogs inaccessible by other means. Discovery of several lead candidates for a neglected tropical disease is a proof-of-concept of this synthetic approach.

Synthesis of multiply substituted 1,6-dihydropyridines through Cu(I)-catalyzed 6-endo cyclization.

Copper-catalyzed 6-endo cyclization of N-propargylic ß-enaminocarbonyls was developed for the synthesis of oxidation-labile 1,6-dihydropyridines. This synthetic method allows flexible and regio-defined assembly of various substituents at the N1, C2, C3, C4, and C6 positions of 1,6-dihydropyridines under mild conditions.

Heterologous expression of highly reducing polyketide synthase involved in betaenone biosynthesis.

A unique highly reducing polyketide synthase (HR-PKS) with a reductase domain was identified in a betaenone biosynthetic gene cluster. Successful heterologous expression and characterization of the HR-PKS and trans-acting enoyl reductase (ER) provide insights into the core structure formation with a decalin scaffold and allow reconstitution of the betaenone biosynthetic machinery.

Allosteric regulation of epoxide opening cascades by a pair of epoxide hydrolases in monensin biosynthesis.

Multistep catalysis of epoxide hydrolase/cyclase in the epoxide opening cascade is an intriguing issue in polyether biosynthesis. A pair of structurally homologous epoxide hydrolases was found in gene clusters of ionophore polyethers. In the epoxide opening reactions with MonBI and MonBII involved in monensin biosynthesis, we found that MonBII and catalytically inactive MonBI mutant catalyzed two-step reactions of bisepoxide substrate analogue to afford bicyclic product although MonBII alone catalyzed only the first cyclization. The X-ray crystal structure of MonBI dimers suggested the importance of the KSD motif in MonBI/MonBI interaction, which was further supported by gel filtration chromatography of wild-type MonBI and mutant MonBI. The involvement of the KSD motif in heterodimer formation was confirmed by in vitro assay. Direct evidence of MonBI/MonBII interaction was obtained by native mass spectrometry. Its dissociation constant was determined as 2.21 × 10(-5) M by surface plasmon resonance. Our results suggested the involvement of an allosteric regulation mechanism by MonBI/MonBII interaction in monensin skeletal construction.