Origin of Octafluorocyclopentene Polyelectrophilicity.

Octafluorocyclopentene (OFCP) has found utility as a polyelectrophile in substitution cascades that form complex macrocyclic compounds. The Harran group synthesis of macrocyclic polypeptides depends on OFCP as a linker, combining with four different nucleophilic units of a polypeptide. We report a computational investigation of the origins of OFCP reactivity and a rationale for controlled mono-, di-, tri-, and tetrasubstitution of fluoride ions by heteroatomic nucleophiles. The roles of inductive, negative hyperconjugative, and resonance electron-donation by fluoride substituents are explored for the reaction of OFCP, less-fluorinated analogues, and common electrophilic alkenes with several different nucleophiles.

Indolizinylalanine Regioisomers: Tryptophan Isosteres with Bathochromic Fluorescence Emission.

We have developed a high yielding synthesis of indolizine and directly elaborated the molecule into three optically active indolizinylalanine regioisomers. The protocols exploit metal catalyzed coupling of indolizinyl-halides with organozinc reagents derived from carbamoylated iodoalanine esters. The scalable protocols provide products in a form amenable to solid-phase peptide synthesis (SPPS). When incorporated into peptides, the indolizine heterocycle is more basic and markedly less nucleophilic than tryptophan. Its protonated vinylpyridinium form is deeply colored in solution while the neutral heterocycle is highly fluorescent. The fluorescence quantum yield of indolizine exceeds that of indole and aza-indoles in water, suggesting that indolizinylalanines could be powerful optical probes of protein structure and dynamics, functioning as true tryptophan isosteres.

Cascade Synthesis of Fluorinated Spiroheterocyclic Scaffolding for Peptidic Macrobicycles.

Octafluorocyclopentene (OFCP) engages linear, unprotected peptides in polysubstitution cascades that generate complex fluorinated polycycles. The reactions occur in a single flask at 0-25 °C and require no catalysts or heavy metals. OFCP can directly polycyclize linear sequences using native functionality, or fluorospiroheterocyclic intermediates can be intercepted with exogenous nucleophiles. The latter tactic generates molecular hybrids composed of peptides, sugars, lipids, and heterocyclic components. The platform can create stereoisomers of both single- and double-looped macrocycles. Calculations indicate that the latter can mimic diverse protein surface loops. Subsets of the molecules have low energy conformers that shield the polar surface area through intramolecular hydrogen bonding. A significant fraction of OFCP-derived macrocycles tested show moderate to high passive permeability in parallel artificial membrane permeability assays.

Computational generation of an annotated gigalibrary of synthesizable, composite peptidic macrocycles.

Peptidomimetic macrocycles have the potential to regulate challenging therapeutic targets. Structures of this type having precise shapes and drug-like character are particularly coveted, but are relatively difficult to synthesize. Our laboratory has developed robust methods that integrate small-peptide units into designed scaffolds. These methods create macrocycles and embed condensed heterocycles to diversify outcomes and improve pharmacological properties. The hypothetical scope of the methodology is vast and far outpaces the capacity of our experimental format. We now describe a computational rendering of our methodology that creates an in silico three-dimensional library of composite peptidic macrocycles. Our open-source platform, CPMG (Composite Peptide Macrocycle Generator), has algorithmically generated a library of 2,020,794,198 macrocycles that can result from the multistep reaction sequences we have developed. Structures are generated based on predicted site reactivity and filtered on the basis of physical and three-dimensional properties to identify maximally diverse compounds for prioritization. For conformational analyses, we also introduce ConfBuster++, an RDKit port of the open-source software ConfBuster, which allows facile integration with CPMG and ready parallelization for better scalability. Our approach deeply probes ligand space accessible via our synthetic methodology and provides a resource for large-scale virtual screening.

In Search of Small Molecules That Selectively Inhibit MBOAT4.

Ghrelin is a 28-residue peptide hormone produced by stomach P/D1 cells located in oxyntic glands of the fundus mucosa. Post-translational octanoylation of its Ser-3 residue, catalyzed by MBOAT4 (aka ghrelin O-acyl transferase (GOAT)), is essential for the binding of the hormone to its receptor in target tissues. Physiological roles of acyl ghrelin include the regulation of food intake, growth hormone secretion from the pituitary, and inhibition of insulin secretion from the pancreas. Here, we describe a medicinal chemistry campaign that led to the identification of small lipopeptidomimetics that inhibit GOAT in vitro. These molecules compete directly for substrate binding. We further describe the synthesis of heterocyclic inhibitors that compete at the acyl coenzyme A binding site.

Photorearrangement of [8]-2,6-Pyridinophane N-Oxide.

Reaction pathways operative when pyridinophane N-oxides are photoirradiated have been studied using time course analyses and careful isolation of photolabile intermediates with support from DFT calculations. Based on the data and the isolation of two previously unknown heterocyclophanes, we outline a unified mechanistic scheme that explains competing processes under varying photochemical conditions.

Callyspongiolide Is a Potent Inhibitor of the Vacuolar ATPase.

Callyspongiolide is a marine-derived macrolide that kills cells in a caspase-independent manner. NCI COMPARE analysis of human tumor cell line toxicity data for synthetic callyspongiolide indicated that its pattern of cytotoxicity correlated with that seen for concanamycin A, an inhibitor of the vacuolar-type H+-ATPase (V-ATPase). Using yeast as a model system, we report that treatment with synthetic callyspongiolide phenocopied a loss of V-ATPase activity including (1) inability to grow on a nonfermentable carbon source, (2) rescue of cell growth via supplementation with Fe2+, (3) pH-sensitive growth, and (4) a vacuolar acidification defect visualized using the fluorescent dye quinacrine. Crucially, in an in vitro assay, callyspongiolide was found to dose-dependently inhibit yeast V-ATPase (IC50 = 10 nM). Together, these data identify callyspongiolide as a new and highly potent V-ATPase inhibitor. Notably, callyspongiolide is the first V-ATPase inhibitor known to be expelled by Pdr5p.

Syntheses of Atypically Fluorinated Peptidyl Macrocycles through Sequential Vinylic Substitutions.

Small peptides containing combinations of cysteine, tyrosine, histidine, and serine residues react with octafluorocyclopentene (OFCP) to afford atypically structured macrocycles through successive vinylic substitutions. The reactions proceed rapidly in air at 0 °C and are tolerant of spectating tryptophan, asparagine, glutamine, and threonine residues. Hexapeptides of consensus sequence YXCXXC displace four fluorine atoms from OFCP to generate fluorinated macrobicyclic compounds that display dual-turn surfaces. The method provides facile access to a wide range of previously unknown heterocyclic structures.

Anomalous Chromophore Disruption Enables an Eight-Step Synthesis and Stereochemical Reassignment of (+)-Marineosin A.

An eight-step asymmetric synthesis of (+)-marineosin A is described. The route proceeds by condensing fragments of reversed polarity relative to conventional prodiginine constructions. The resultant unstable chromophore is disrupted by a unique cycloisomerization promoted at a tailored manganese surface. This provides a premarineosin and subsequently marineosin A in a particularly concise manner. A pyridinophane N-oxide photorearrangement in flow and structural isomers of premarineosin are discussed, as is the reassignment of marineosin stereochemistry. The route gives access to the natural product as well as diastereomers, congeners and analogs that are currently inaccessible by other means.

Exploring the Boundaries of "Practical": De Novo Syntheses of Complex Natural Product-Based Drug Candidates.

This review examines the state of the art in synthesis as it relates to the building of complex architectures on scales sufficient to drive human drug trials. We focus on the relatively few instances in which a natural-product-based development candidate has been manufactured de novo, rather than semisynthetically. This summary provides a view of the strengths and weaknesses of current technologies, provides perspective on what one might consider a practical contribution, and hints at directions the field might take in the future.

Unconventional Fragment Usage Enables a Concise Total Synthesis of (-)-Callyspongiolide.

An asymmetric synthesis of (-)-callyspongiolide is described. The route builds the macrolide domain atypically from a disaccharide and a monoterpene without passing through a seco-acid. Chiral iridium catalysis selectively joins fragments. Subsequent degradation of an imbedded butyrolactone via perhemiketal fragmentation affords a stereo- and regio-defined homoallylic alcohol that is engaged directly in a carbonylative macrolactonization. Further elaboration of the polyunsaturated appendage provides the natural product in a particularly direct and flexible manner.

Cascading Auto-oxidative Biproline Guanylations Form Optically Active Dispacamide Dimers and Permit an Eight-Step Synthesis of (-)-Ageliferin.

A nickel catalyzed synthesis of isomeric 3,3'-biproline esters is described. When those materials are doubly acylated with the acid chloride of pyrrole-2-carboxylic acid, they become susceptible to auto-oxidation in the presence of guanidine. Through proper staging of reaction conditions, it is possible to initiate two consecutive oxidative guanylations prior to in situ cycloisomerization to afford spirocyclic bis-glycocyamidines. This unique outcome reflects a cascade of no fewer than 10 reactions occurring sequentially in one flask. The chemistry provides rapid access to advanced intermediates useful for the preparation of complex, optically active pyrrole/imidazole alkaloids.

Assembly of Complex Macrocycles by Incrementally Amalgamating Unprotected Peptides with a Designed Four-Armed Insert.

We describe the asymmetric synthesis of a highly substituted ω-octynoic acid derivative and demonstrate its utility for generating complex macrocycles from unprotected peptides. The molecule harbors an isolated quaternary center that displays four uniquely functionalized arms, each of which can be reacted orthogonally in sequence as the molecule is integrated into peptide structure. These processing sequences entail (1) scaffold ligation, (2) macrocyclization via internal aromatic alkylations or catalyzed etherifications, (3) acyliminium ion mediated embedding of condensed heterocycles, and (4) terminal alkyne derivatization or dimerization reactions. Numerous polycycles are prepared and fully characterized in this study. Factors that influence reaction efficiencies and selectivity are also probed. We construct a novel mimic of the second mitochondria derived activator of caspase using these techniques, wherein subtle variations in macrocycle connectivity have a marked impact on performance. In general, the chemistry is an important step toward facile, systematic access to complex peptidomimetics synthesized by directly altering the structure and properties of machine-made oligomers.

Template-constrained macrocyclic peptides prepared from native, unprotected precursors.

Peptide-protein interactions are important mediators of cellular-signaling events. Consensus binding motifs (also known as short linear motifs) within these contacts underpin molecular recognition, yet have poor pharmacological properties as discrete species. Here, we present methods to transform intact peptides into stable, templated macrocycles. Two simple steps install the template. The key reaction is a palladium-catalyzed macrocyclization. The catalysis has broad scope and efficiently forms large rings by engaging native peptide functionality including phenols, imidazoles, amines, and carboxylic acids without the necessity of protecting groups. The tunable reactivity of the template gives the process special utility. Defined changes in reaction conditions markedly alter chemoselectivity. In all cases examined, cyclization occurs rapidly and in high yield at room temperature, regardless of peptide composition or chain length. We show that conformational restraints imparted by the template stabilize secondary structure and enhance proteolytic stability in vitro. Palladium-catalyzed internal cinnamylation is a strong complement to existing methods for peptide modification.

Tailored fragments of roseophilin selectively antagonize Mcl-1 in vitro.

We have discovered a fragment of the natural product roseophilin, a member of the prodiginine family, that antagonizes Mcl-1 functions in a liposome-based assay for mitochondrial membrane permeabilization. By tailoring this substance such that it can participate in salt bridging with the protein surface, we have prepared the first prodiginine inspired structure that shows direct, saturable binding to a recombinant Bcl-2 family member in vitro.

Electrolytic macrocyclizations: scalable synthesis of a diazonamide-based drug development candidate.

An electrochemical method to synthesize the core macrolactam of diazonamides is described. Large ring-forming dehydrogenation is initiated by anodic oxidation at a graphite surface. The reaction requires no tailoring of the substrate and occurs at ambient temperature in aqueous DMF in an undivided cell open to air. This unique chemistry has enabled a concise, scalable preparation of DZ-2384; a refined analog of diazonamide A slated for clinical development as a cancer therapeutic.

Modular access to complex prodiginines: total synthesis of (+)-roseophilin via its 2-azafulvene prototropisomer.

Ansa-bridged prodiginines are bioactive pigments produced by bacteria. Certain of these structures are reported to be antagonists of protein-protein interactions involved in apoptosis. We describe a new entry to alkaloids of this type, demonstrated with a concise asymmetric synthesis of (+)-roseophilin (3). Our route constructs the pyrrolophane motif via phosphoryl transfer-terminated macroaldolization and passes through a previously unexplored prototropic form of the natural product.

Template-induced macrocycle diversity through large ring-forming alkylations of tryptophan.

Macrocyclic peptidomimetics are valuable in research and serve as lead compounds in drug discovery efforts. New methods to prepare such structures are of considerable interest. In this pilot study, we show that an organic template harboring a latent cinnamyl cation participates in novel Friedel-Crafts macrocyclization reactions with tryptophan. Upon joining the template to Trp-Trp-Tyr, a single operation efficiently generates eight unique macrocycles. Each has been isolated and thoroughly characterized. Product distribution as a function of Brønsted and/or Lewis acidic conditions was explored, and outcomes were compared to rearrangements induced within a corresponding tyrosine-linked cyclic ether. The solution structure of a new macrocyclic pyrroloindoline was solved using a combination of two-dimensional NMR methods and molecular mechanics simulations. Template-induced structural diversification of peptide sequences harboring aromatic residues has potential to create myriad macrocycles that target surfaces involved in protein-protein interactions.

Substituted 2,2'-bipyrroles and pyrrolylfurans via intermediate isoxazolylpyrroles.

We describe a new synthesis of the 3-chloro-(4'-methoxy)-2,2'-pyrrolylfuran segment (3) of (+)- roseophilin. The route exploits a isoxazoylpyrrole intermediate, wherein the isoxazole ring serves as a ß-diketone equivalent and a directing group for palladium catalyzed chlorination of the attached pyrrole. Subsequent reduction of the N-O bond and acid promoted cyclization afords roseophilin segment 3b in five steps and 19% overall yield. This strategy was extended to the synthesis of 3-chloro-(4'-alkoxy)-2,2'-pyrrolylfurans (16a-c) and 4-alkoxy-2,2'-bipyrroles (20a-c), which are building blocks to synthesize bioactive prodiginine natural products and their congeners.

Asymmetric Organocatalysis Enables Rapid Assembly of Portimine Precursor Chains.

Sequential organocatalytic additions of 2-furanone and dihydroxyacetone derivatives to a crotonaldehyde lynchpin provide polyhydroxylated chains reminiscent of lactonized deoxo Kdn type sugars. Further homologation via Kulinkovich ring opening of the butyrolactone and acylation of the zinc homoenolate derived from the incipient cyclopropanol allows assembly of functionalized chain precursors to portimine. Our experiments probe the stability and reactivity of monosubstituted methylidene pyrrolines and generate advanced intermediates useful for exploring the biosynthesis and de novo synthesis of portimine.