Albert Eschenmoser (1925-2023).

Organic chemist who demystified the logic of natural product structures.

Exploring the Chemical Space of Proluciferins as Probe Substrates for Human Cytochrome P450 Enzymes.

We report the synthesis of 21 new proluciferin compounds that bear a small aliphatic ether group connected to the 6' hydroxy function of firefly luciferin and either contain an acid or methyl ester function at the dihydrothiazole ring. Each of these compounds was found to be a substrate for some members of the human CYP1 and CYP3 families; a total of 92 new enzyme-substrate pairs were identified. In a screen of the whole human P450 complement (CYPome) with three selected proluciferin acid substrates, another 13 enzyme-substrate pairs were detected, which involve enzymes belonging to the CYP2, CYP4, CYP7, CYP21, and CYP27 families. All in all, we identified new probe substrates for members of seven out of 18 human CYP families.

A Concise Synthesis of Pleurotin Enabled by a Nontraditional C-H Epimerization.

An 8-step synthesis of a known pentacyclic intermediate toward the natural product pleurotin (1) is described. Pleurotin and related benzoquinone natural products are of great interest for their powerful anticancer and antibiotic activities. The route features a regio- and diastereoselective intermolecular photoenolization/Diels-Alder cycloaddition and an alkoxy-radical-induced hydrogen atom transfer-mediated C-H epimerization to construct pleurotin's carbon framework with appropriate relative stereochemical relationships. The synthesis concludes with a ring-forming benzylic C-H oxidation to deliver oxepane 19.

A C-H Functionalization Strategy Enables an Enantioselective Formal Synthesis of (-)-Aflatoxin B2.

An enantioselective formal synthesis of (-)-aflatoxin B2 from 4-methoxyphenylacetic acid has been achieved by an approach that produces a key carbon-carbon bond, a benzylic stereocenter, and two arene carbon-oxygen bonds in the course of three site-selective C-H functionalizations. The carbonyl-directed acetoxylation of two arene C-H bonds described herein is unprecedented in natural product synthesis and occurs under mild conditions that preserve the configuration of a sensitive benzylic stereocenter.

Pd(II)-Catalyzed Synthesis of Benzocyclobutenes by ß-Methylene-Selective C(sp3)-H Arylation with a Transient Directing Group.

Methylene-selective C-H functionalization is a significant hurdle that remains to be addressed in the field of Pd(II) catalysis. We report a Pd(II)-catalyzed synthesis of benzocyclobutenes by methylene-selective C(sp3)-H arylation of ketones. The reaction utilizes glycine as a transient directing group and a 2-pyridone ligand, which may govern the methylene selectivity by making intimate molecular associations with the substrate during concerted metalation-deprotonation. This reaction is shown to be highly selective for intramolecular methylene C(sp3)-H arylation, thus enabling sequential C(sp3)-H functionalization.

Conversion of five proluciferin esters by human cytochrome P450 enzymes.

BACKGROUND: Probe substrates are an important tool for activity monitoring of human drug metabolizing enzymes such as cytochromes P450 (CYPs). BRIEF METHODS: In the present study we have tested human CYPs for metabolization of five proluciferin ester substrates which had previously only been known to be hydroxylated by CYP26A1. MAJOR RESULTS: It was found that these substrates were converted by another 21 human CYPs, which belong to the CYP families 1 to 4, 7, and 26. Thus, 66 new pairs of enzyme and substrate were identified. Correlation analysis indicated the presence of three distinct sets of enzymes with high similarity in their activity profiles that encompass a total of 16 individual enzymes. CONCLUSIONS: Some of these newly identified correlations may serve as a starting point for further study of those human CYPs whose activities are not yet satisfactorily understood.

New Proluciferin Substrates for Human CYP4 Family Enzymes.

We report the synthesis of seven new proluciferins for convenient activity determination of enzymes belonging to the cytochrome P450 (CYP) 4 family. Biotransformation of these probe substrates was monitored using each of the twelve human CYP4 family members, and eight were found to act at least on one of them. For all substrates, activity of CYP4Z1 was always highest, while that of CYP4F8 was always second highest. Site of metabolism (SOM) predictions involving SMARTCyp and docking experiments helped to rationalize the observed activity trends linked to substrate accessibility and reactivity. We further report the first homology model of CYP4F8 including suggested substrate recognition residues in a catalytically competent conformation accessed by replica exchange solute tempering (REST) simulations.

New luciferin-based probe substrates for human CYP26A1.

Activity of human CYP26A1 towards six proluciferin probe substrates and their ester derivatives was monitored. These included three monofluorobenzyl ether isomers and three five-membered heterocycles. Overall, luciferin substrates with a free acid group gave higher activities than the ester compounds. Also, luciferin derivatives with six-ring structures were better metabolized than those with five-rings. The best substrates identified in this study are Luciferin 6' 3-fluorobenzyl ether (Luciferin-3FBE) and its methyl ester (Luciferin-3FBEME). Taken together, we describe eleven new probe substrates for CYP26A1 and demonstrate for the first time that CYP26A1 does not only accept acid substrates but can also metabolize esters.

Donor-Acceptor-Acceptor 1,3-Bisdiazo Compounds: An Exploration of Synthesis and Stepwise Reactivity.

Metal carbenes, derived from the decomposition of diazo compounds, are valued for their capacity to perform a variety of transformations. A unique class of acyclic, bis-diazo compounds, the donor-acceptor-acceptor 1,3-bisdiazo compounds, are described herein. These compounds are available from acyclic ß-keto esters and especially reactive at the donor-acceptor diazo unit. These bisdiazo compounds react smoothly with rhodium acetate and alcohols to give monodiazo, cyclic orthoesters, presumably through the capture of a transient oxonium ylide.

Importance of asparagine-381 and arginine-487 for substrate recognition in CYP4Z1.

The human cytochrome P450 enzyme CYP4Z1 remains an understudied enzyme despite its association with poor prognosis and overexpression in breast cancer. Hence, CYP4Z1 has previously been suggested as an anti-breast cancer target. In the present study we employed extended mutation analysis to increase our understanding of the substrate binding mode of this enzyme. In a combined in vitro and in silico approach we show for the first time that residue Arg487 plays an important role in substrate recognition and binding of CYP4Z1. Using a large array of recombinant CYP4Z1 mutants we show that, apart from Asn381, all other postulated binding residues only play an auxiliary role in substrate recognition and binding. Different substrate interaction motifs were identified via dynamic pharmacophores (dynophores) and their impact on catalytically competent substrate binding was classified. These new insights on the substrate recognition and binding mode represent an important step towards the rational design of CYP4Z1 prodrugs and guide further investigations into the so far poorly understood physiological role of CYP4Z1.

Synthesis of (+)-Lineariifolianone and Related Cyclopropenone-Containing Sesquiterpenoids.

A synthesis of the proposed structure of lineariifolianone has been achieved in eight steps and 9% overall yield starting from (+)-valencene, leading to a reassignment of the absolute configuration of this unusual cyclopropenone-containing natural product. Key steps in the synthetic route include kinetic protonation of an enolate to epimerize the C7 stereocenter and a stereoconvergent epoxide opening to establish the trans-diaxial diol functionality. The syntheses of the enantiomers of two other closely related natural products are also reported, confirming that all three compounds belong to the eremophilane class of sesquiterpenoids.

Crystal Structure of the Cyclostreptin-Tubulin Adduct: Implications for Tubulin Activation by Taxane-Site Ligands.

It has been proposed that one of the mechanisms of taxane-site ligand-mediated tubulin activation is modulation of the structure of a switch element (the M-loop) from a disordered form in dimeric tubulin to a folded helical structure in microtubules. Here, we used covalent taxane-site ligands, including cyclostreptin, to gain further insight into this mechanism. The crystal structure of cyclostreptin-bound tubulin reveals covalent binding to ßHis229, but no stabilization of the M-loop. The capacity of cyclostreptin to induce microtubule assembly compared to other covalent taxane-site agents demonstrates that the induction of tubulin assembly is not strictly dependent on M-loop stabilization. We further demonstrate that most covalent taxane-site ligands are able to partially overcome drug resistance mediated by ßIII-tubulin (ßIII) overexpression in HeLa cells, and compare their activities to pironetin, an interfacial covalent inhibitor of tubulin assembly that displays invariant growth inhibition in these cells. Our findings suggest a relationship between a diminished interaction of taxane-site ligands with ßIII-tubulin and ßIII tubulin-mediated drug resistance. This supports the idea that overexpression of ßIII increases microtubule dynamicity by counteracting the enhanced microtubule stability promoted by covalent taxane-site binding ligands.

Recent applications of C-H functionalization in complex natural product synthesis.

In this review, recent examples featuring C-H functionalization in the synthesis of complex natural products are discussed. A focus is given to the way in which C-H functionalization can influence the logical process of retrosynthesis, and the review is organized by the type and method of C-H functionalization.

Installation of Multiple Aryl Ether Crosslinks onto Non-Native Substrate Peptides by the Vancomycin OxyB.

The biosynthesis of glycopeptide antibiotics (GPAs) has been an active area of research for decades. Nonetheless, insights into the activity of the cytochrome P450 enzymes required for installing the aromatic crosslinks, which form their cup-shaped topologies and render GPAs bioactive, have only recently emerged. Presently, little is known about the substrate scope and promiscuity of the P450 enzymes. Herein, we report that OxyBvan, the P450 enzyme that installs the first crosslink in vancomycin biosynthesis, is capable of catalyzing the formation of its conventional C-O-D bis-aryl ether bond in non-natural substrates and, furthermore, the formation of a second, novel linkage when D-Trp is incorporated at position 6. HR-MS/MS and isotope labeling studies indicate the second crosslink is formed between rings A and B, resulting in a novel GPA-type scaffold. OxyB is also capable of installing two crosslinks in kistamicin- and complestatin-like substrate peptides. These findings highlight the utility of OxyBvan in creating crosslinked GPA derivatives and provide clues regarding the unusual biosynthesis of kistamicin.

Modulating OxyB-Catalyzed Cross-Coupling Reactions in Vancomycin Biosynthesis by Incorporation of Diverse d-Tyr Analogues.

We report a general method for synthesizing diverse d-Tyr analogues, one of the constituents of the antibiotic vancomycin, using a Negishi cross-coupling protocol. Several analogues were incorporated into the vancomycin substrate-peptide and reacted with the biosynthetic enzymes OxyB and OxyA, which install the characteristic aromatic cross-links. We find that even small structural perturbations are not accepted by OxyA. The same modifications, however, enhance the catalytic capabilities of OxyB leading to the formation of a new macrocycle within the vancomycin framework.

Pd-Catalyzed, ortho C-H Methylation and Fluorination of Benzaldehydes Using Orthanilic Acids as Transient Directing Groups.

The direct, Pd-catalyzed ortho C-H methylation and fluorination of benzaldehydes have been accomplished using commercially available orthanilic acids as transient directing groups. In these reactions, the 1-fluoro-2,4,6-trimethylpyridinium salts can be either a bystanding F+ oxidant or an electrophilic fluorinating reagent. An X-ray crystal structure of a benzaldehyde ortho C-H palladation intermediate was obtained using triphenylphosphine as the stabilizing ligand.

Ir(iii)-catalyzed ortho C-H alkylations of (hetero)aromatic aldehydes using alkyl boron reagents.

Transition-metal-catalyzed C-H alkylation reactions directed by aldehydes or ketones have been largely restricted to electronically activated alkenes. Herein, we report a general protocol for the Ir(iii)-catalyzed ortho C-H alkylations of (hetero)aromatic aldehydes using alkyl boron reagents as the coupling partner. Featuring aniline as an inexpensive catalytic ligand, the method was compatible with a wide variety of benzaldehydes, heterocyclic aldehydes, potassium alkyltrifluoroborates as well as a few α,ß-unsaturated aldehydes. An X-ray crystal structure of a benzaldehyde ortho C-H iridation intermediate was also successfully obtained.

Pd-Catalyzed Ortho C-H Hydroxylation of Benzaldehydes Using a Transient Directing Group.

The direct Pd-catalyzed ortho C-H hydroxylation of benzaldehydes was achieved using 4-chloroanthranilic acid as the transient directing group, 1-fluoro-2,4,6-trimethylpyridnium triflate as the bystanding oxidant, and p-toluenesulfonic acid as the putative oxygen nucleophile. The unusual C-H chlorination and polyfluoroalkoxylation reactions signaled the importance of external nucleophiles to the outcome of Pd(IV) reductive eliminations.

Efficient substrate screening and inhibitor testing of human CYP4Z1 using permeabilized recombinant fission yeast.

We have established a protocol for the preparation of permeabilized fission yeast cells (enzyme bags) that recombinantly express human cytochrome P450 enzymes (CYPs). A direct comparison of CYP3A4 activity gave an eightfold higher space-time yield for enzyme bag-catalyzed biotransformation as compared to whole-cell biotransformation, even though the total number of cells employed was lower by a factor of 150. Biotransformation of the luminogenic substrate Luciferin-H using CYP2C9-containing enzyme bags proceeded efficiently and stably for 24h. CYP4Z1 is of interest because it is strongly overexpressed both in breast cancer cells and in breast cancer metastases; however, current knowledge about its catalytic properties is very limited. Screening of CYP4Z1-containing enzyme bags with 15 luminogenic substrates enabled us to identify two new hydroxylations and eleven ether cleavage reactions that are catalyzed by CYP4Z1. By far the best substrate found in this study was Luciferin benzyl ether (Luciferin-BE). On the basis of the recently published crystal structure of CYP4B1 we created a new homology model of CYP4Z1 and performed molecular docking experiments, which indicate that all active substrates show a highly similar binding geometry compared to the endogenous substrates. The model predicts that Ser113, Ser222, Asn381, and Ser383 are key hydrogen bonding residues. We also identified five new inhibitors of CYP4Z1: miconazole, econazole, aminobenzotriazole, tolazoline, and 1-benzylimidazole respectively, with the last compound being the most potent giving an IC50 value of 180nM in our test system.

A Stereocontrolled Annulation of the Taccalonolide Epoxy Lactone onto the Molecular Framework of trans-Androsterone.

A robust and scalable route to the taccalonolide skeleton starting from trans-androsterone is presented. The synthesis features a cyclic hydroboration carbonylation reaction, which effectively establishes the trans-hydrindane DE ring junction in a remarkable annulation reaction, as well as a Claisen rearrangement and a catalytic Ullmann-type cyclization. This work is part of a larger effort to uncover new clinical candidates from the taccalonolide class of anticancer agents through advances in chemical synthesis.