Alkyne Moiety Research Articles

Discovery of a Dual Function Cytochrome P450 that Catalyzes Enyne Formation in Cyclohexanoid Terpenoid Biosynthesis

AbstractThe 1,3‐enyne moiety is commonly found in cyclohexanoid natural products produced by endophytic and plant pathogenic fungi. Asperpentyn (1) is a 1,3‐enyne‐containing cyclohexanoid terpenoid isolated from Aspergillus and Pestalotiopsis. The genetic basis and biochemical mechanism of 1,3‐enyne biosynthesis in 1, and other natural products containing this motif, has remained enigmatic despite their potential ecological roles. Identified here is the biosynthetic gene cluster and characterization of two crucial enzymes in the biosynthesis of 1. A P450 monooxygenase that has a dual function, to first catalyze dehydrogenation of the prenyl chain to generate a cis‐diene intermediate and then serve as an acetylenase to yield an alkyne moiety, and thus the 1,3‐enyne, was discovered. A UbiA prenyltransferase was also characterized and it is unusual in that it favors transferring a five‐carbon prenyl chain, rather than a polyprenyl chain, to a p‐hydroxybenzoic acid acceptor.

Discovery of a Dual Function Cytochrome P450 that Catalyzes Enyne Formation in Cyclohexanoid Terpenoid Biosynthesis.

The 1,3-enyne moiety is commonly found in cyclohexanoid natural products produced by endophytic and plant pathogenic fungi. Asperpentyn (1) is a 1,3-enyne-containing cyclohexanoid terpenoid isolated from Aspergillus and Pestalotiopsis. The genetic basis and biochemical mechanism of 1,3-enyne biosynthesis in 1, and other natural products containing this motif, has remained enigmatic despite their potential ecological roles. Identified here is the biosynthetic gene cluster and characterization of two crucial enzymes in the biosynthesis of 1. A P450 monooxygenase that has a dual function, to first catalyze dehydrogenation of the prenyl chain to generate a cis-diene intermediate and then serve as an acetylenase to yield an alkyne moiety, and thus the 1,3-enyne, was discovered. A UbiA prenyltransferase was also characterized and it is unusual in that it favors transferring a five-carbon prenyl chain, rather than a polyprenyl chain, to a p-hydroxybenzoic acid acceptor.

Ruthenium-Catalyzed Propargylic Reduction of Propargylic Alcohols with Hantzsch Ester

Ruthenium-catalyzed propargylic reduction of propargylic alcohols bearing a terminal alkyne moiety is accomplished by using Hantzsch ester as a nucleophilic hydride source. A variety of secondary a...

Recent Strides in the Transition Metal-Free Cross-Coupling of Haloacetylenes with Electron-Rich Heterocycles in Solid Media.

The publications covering new, transition metal-free cross-coupling reactions of pyrroles with electrophilic haloacetylenes in solid medium of metal oxides and salts to regioselectively afford 2-ethynylpyrroles are discussed. The reactions proceed at room temperature without catalyst and base under solvent-free conditions. These ethynylation reactions seem to be particularly important, since the common Sonogashira coupling does not allow ethynylpyrroles with strong electron-withdrawing substituents at the acetylenic fragments to be synthesized. The results on the behavior of furans, thiophenes, and pyrazoles under the conditions of these reactions are also provided. The reactivity and structural peculiarities of nucleophilic addition to the activated acetylene moiety of the novel C-ethynylpyrroles are considered.

Biosynthesis of Biscognienyne B Involving a Cytochrome P450‐Dependent Alkynylation

AbstractThe alkyne is a biologically significant moiety found in many natural products and a versatile functional group widely used in modern chemistry. Recent studies have revealed the biosynthesis of acetylenic bonds in fatty acids and amino acids. However, the molecular basis for the alkynyl moiety in acetylenic prenyl chains occurring in a number of meroterpenoids remains obscure. Here, we identify the biosynthetic gene cluster and characterize the biosynthetic pathway of an acetylenic meroterpenoid biscognienyne B based on heterologous expression, feeding experiments, and in vitro assay. This work shows that the alkyne moiety is constructed by an unprecedented cytochrome P450 enzyme BisI, which shows promiscuous activity towards C5 and C15 prenyl chains. This finding provides an opportunity for discovery of new compounds, featuring acetylenic prenyl chains, through genome mining, and it also expands the enzyme inventory for de novo biosynthesis of alkynes.

Biosynthesis of Biscognienyne B Involving a Cytochrome P450-Dependent Alkynylation.

The alkyne is a biologically significant moiety found in many natural products and a versatile functional group widely used in modern chemistry. Recent studies have revealed the biosynthesis of acetylenic bonds in fatty acids and amino acids. However, the molecular basis for the alkynyl moiety in acetylenic prenyl chains occurring in a number of meroterpenoids remains obscure. Here, we identify the biosynthetic gene cluster and characterize the biosynthetic pathway of an acetylenic meroterpenoid biscognienyne B based on heterologous expression, feeding experiments, and in vitro assay. This work shows that the alkyne moiety is constructed by an unprecedented cytochrome P450 enzyme BisI, which shows promiscuous activity towards C5 and C15 prenyl chains. This finding provides an opportunity for discovery of new compounds, featuring acetylenic prenyl chains, through genome mining, and it also expands the enzyme inventory for de novo biosynthesis of alkynes.

Biological macromolecule binding and anticancer activity of synthetic alkyne-containing L-phenylalanine derivatives.

Herein, we described the synthesis of two L-phenylalanines α-derivatized with a terminal alkyne moiety whose structures differed by phenyl ring halogen substitution (two o-Cl in 1 vs. one p-Br in 2) and investigated their effect on biological macromolecules and living cells. We explored their interaction with quadruplex DNA (G4 DNA), using tel26 and c-myc as models, and bovine serum albumin (BSA). By CD spectroscopy, we found that 1 caused minor tel26 secondary structure changes, leading also to a slight thermal stabilization of this hybrid antiparallel/parallel G4 structure, while the c-myc parallel topology remained essentially unchanged upon 1 binding. Other CD evidences showed the ability of 1 to bind BSA, while molecular docking studies suggested that the same molecule could be housed into the hydrophobic cavity between sub-domains IIA, IIB, and IIIA of the protein. Furthermore, preliminary aggregation studies, based on concentration-dependent spectroscopic experiments, suggested the ability of 1 to aggregate forming noncovalent polymeric systems in aqueous solution. Differently from 1, the bromine-modified compound was able to bind Cu(II) ion, likely with the formation of a CuL2 complex, as found by UV spectroscopy. Finally, cell tests excluded anycytotoxic effect of both compounds toward normal cells, but showed slight antiproliferative effects of 2 on PC3 cancerous cells at 24h, and of 1 on both T98G and MDA-MB-231 cancer cells at 48h.

Synthesis of hybrid perillyl‐4H‐pyrans. Cytotoxicity evaluation against hepatocellular carcinoma HepG2/C3A cell line

AbstractA series of 15 new hybrid perillyl‐4H‐pyrans compounds was straightforwardly synthesized by a strategy combining the multicomponent reaction and the copper‐catalyzed alkyne‐azide cycloaddition (CuAAC). The 2‐amino‐4H‐pyrans‐3‐carbonitrile containing the alkyne moiety was prepared via multicomponent reaction between 1,3‐dicarbonyl, a propargyloxy aromatic aldehyde and malononitrile or ethyl α‐cyanoacetate. The alkyne derivative was sequentially reacted with the perillyl azide component through the copper‐catalyzed [3+2] Huisgen cycloaddition reaction. The antiproliferative activity of hybrid compounds were evaluated against the human hepatoma HepG2/C3A cell line.

Optimization of Propargylcholine to Label Newly Synthesized Myelin in the Rhesus Monkey Brain

Neuropathology in monkey and human brains often involves loss or damage to myelin and is a major factor impacting normal function as myelin constitutes 35%–40% of the forebrain. Methods to assess alterations in myelin include electron microscopy, histological stains (e.g. Luxol Fast Blue), immunohistochemistry (e.g. for myelin basic protein) and a label free confocal method (SCoRe), as well as diffusion MRI. Each method has advantages but none offer a reliable, validated method to assess microscopic changes in myelin (synthesis or repair) across the entire forebrain from gray matter fascicles to subcortical white matter. Here we present preliminary data that newly synthesized myelin can be detected with in vivo administration of a choline analog, propargylcholine (P‐Cho), that is readily incorporated into all classes of choline phospholipids including major components of the myelin sheath, phosphatidylcholine and sphingomyelin. Importantly, since P‐Cho contains an alkyne moiety, it can be visualized post mortem by conjugation with a fluoro‐labeled azide using click chemistry and detection with confocal microscopy. P‐Cho can be administered by intraperitoneal injection where it enters the circulation and is incorporated into myelin, allowing detection and quantification of newly synthesized myelin. While previous research has demonstrated the use of P‐Cho for assessing remyelination in mice, its efficacy and safety in monkeys has yet to be explored. This is important for our monkey model of normal aging where age‐related myelin pathology accounts for cognitive deficits and for our studies of recovery after damage to the motor cortex. The ability to quantify myelin plasticity in both of these models could provide significant insight into myelin pathology, repair, and maintenance. To test this, we administered P‐Cho daily for 6 days via IP injections (3.5 – 4.0 mg/kg) to 6 young adult and 1 older adult monkey at different time points before euthanasia (1, 2 and 6 weeks). Frozen brain sections were cut, labeled with a fluorescent azide, and the sections evaluated for the presence and specificity of P‐Cho by co‐labeling the tissue with antibodies for axons (SMI‐312) and newly formed actively myelinating oligodendrocytes (BCAS1). Co‐localization of P‐Cho was also performed using confocal reflectance microscopy (SCoRe). Preliminary analyses suggest effective incorporation of P‐Cho into newly synthesized myelin within 1 week of dosing and sustained presence beyond 6 weeks. These results demonstrate a promising new method for investigating remyelination and white matter pathologies related to aging that could also be applied to studies of neurodegeneration, brain injury, and neurological diseases in an animal model highly translatable to humans.Support or Funding InformationNIH Grants R01‐AG062831, R01‐AG043640; R01‐AG042512; R21‐AG061678; R21‐NS111174, U01‐NS076474

Design, Synthesis and Characterization of Novel Isoxazole Tagged Indole Hybrid Compounds

AbstractSixteen new isoxazole tagged indole compounds have been synthesized via copper (I) catalyzed click chemistry of the aryl hydroxamoyl chloride and an indole containing alkyne moiety. The chemical structure of the synthesized compounds has been established using various physicochemical techniques. X-ray single crystal analysis of Ethyl 1-((3-phenylisoxazol-5-yl) methyl)-1H-indole-2-carboxylate (8a) has been analyzed. All compounds were tested for their antibacterial and anticancer activities. The activities for the new compounds were weak against both bacterial strains and the cancer cell lines.

Optimizing the Biodistribution of Radiofluorinated Barbiturate Tracers for Matrix Metalloproteinase Imaging by Introduction of Fluorescent Dyes as Pharmacokinetic Modulators.

Dysregulated expression or activation of matrix metalloproteinases (MMPs) is observed in many kinds of life-threatening diseases. Therefore, MMP imaging-for example, with radiolabeled MMP inhibitors (MMPIs)-potentially represents a valuable tool for clinical diagnostics using noninvasive single photon emission computed tomography (SPECT) or positron emission tomography (PET) imaging. Despite numerous preclinical imaging approaches, translation to a clinical setting has not yet been successful. We introduce and oppose three potential radiofluorinated MMP-targeted imaging probes, modified by the introduction of pentamethine cyanine (Cy5) dyes and therefore containing both radio- as well as fluorescent label with respect to their capability to assess MMP activity in vivo by means of scintigraphic (PET) and/or fluorescent (NIRF) imaging. New hybrid MMPI tracer candidates, structurally based on radiofluorinated pyrimidine-2,4,6-triones (barbiturates) from previous approaches, were synthesized by convenient two-step syntheses. In the first step, Cy5 dyes, varying in the number of sulfonate groups (nSO3- = 1, 2, or 4) and bearing an additional "clickable" alkyne moiety, were coupled to the barbiturate MMPI by amide formation. In the second step, the [18F]fluoride radiolabel was introduced into the resulting Cy5 dye conjugates by "radio-click" chemistry. Biodistribution studies of these hybrid tracer candidates were assessed and compared in C57BL/6 mice by PET as well as fluorescence imaging. MMP activity was imaged in a MMP-positive mouse model of irritant contact dermatitis (ICD) by PET and sequential fluorescence reflectance imaging (FRI), respectively. In vivo data were validated by scintillation counting, gelatin zymography, and MMP-histology. Three new potential hybrid MMP imaging probes were prepared, differing essentially in the number of sulfonate groups, introduced by Cy5 dye components. Although the hydrophilicity of these compounds was substantially increased, 10a (nSO3- = 1) and 10b (nSO3- = 2) were still rapidly eliminated via unfavorable hepatobiliary pathways, as observed in earlier approaches. Only 11 (nSO3- = 4) showed delayed in vivo clearance and a shift towards higher renal elimination. In the chosen mouse model of ICD, only 11 (nSO3- = 4) significantly accumulated in the inflamed mouse ear, which could be precisely visualized by means of PET and FRI.

Dual C‐C Bond Forming Heck and Sonogashira Couplings Followed by Hydroarylation: Synthesis of Tricyclic Frameworks

AbstractHerein, a concise approach to construct the novel tricyclic ether frameworks, is presented. Microwave‐assisted dual C‐C bond‐forming palladium‐catalyzed intramolecular Heck/intermolecular Sonogashira couplings were implemented, for the synthesis of dihydrobenfurans bearing acetylene moiety, followed by acid‐induced intramolecular hydroarylation reaction of the crude reaction mixture furnished the tricyclic ether systems. Notably, these tricyclic ether frameworks have been achieved, in excellent yields, through a single column chromatography process.

Chemically labeled toxins or bioactive peptides show a heterogeneous intracellular distribution and low spatial overlap with autofluorescence in bloom-forming cyanobacteria

Harmful algal blooms formed by colony-forming cyanobacteria deteriorate water resources by producing cyanotoxins, which frequently occur at high intracellular concentrations. We aimed to localize toxic microcystins (MCs) and bioactive anabaenopeptins (APs) at the subcellular level under noninvasive conditions. Since both metabolites are synthesized nonribosomally, the relaxed specificity of key enzymes catalyzing substrate activation allowed chemical labeling through a standard copper-catalyzed click chemistry reaction. The genera Planktothrix and Microcystis specifically incorporated unnatural amino acids such as N-propargyloxy-carbonyl-L-lysine or O-propargyl-L-tyrosine, resulting in modified AP or MC peptides carrying the incorporated alkyne moiety. The labeled cells were quantitatively differentiated from the unlabeled control cells. MCs and APs occurred intracellularly as distinct entities showing a cell-wide distribution but a lowered spatial overlap with natural autofluorescence. Using the immunofluorescence technique, colocalization with markers of individual organelles was utilized to relate the distribution of labeled MCs to cellular compartments, e.g., using RbcL and FtsZ (cytosol) and PsbA (thylakoids). The colocalization correlation coefficients calculated pairwise between organelles and autofluorescence were highly positive as opposed to the relatively low positive indices derived from labeled MCs. The lower correlation coefficients imply that only a portion of the labeled MC molecules were related spatially to the organelles in the cell.

Carbazole‐Based N‐Heterocyclic Carbenes for the Promotion of Copper‐Catalyzed Palladium‐Free Homo‐/Hetero‐Coupling of Alkynes and Sonogashira Reactions

AbstractAlkynes are useful synthetic building blocks that occur in many naturally occurring molecules and have the potential to be easily modified for further natural products and drug synthesis applications. Given the‐versatility of the alkyne functionality,‐various synthetic methods (mostly palladium‐based) are known in the literature, allowing the installation to take place under different reaction conditions. A single catalytic system (without Pd) however, if developed could provide a single point solution for the installation of the alkyne moiety and keeping this in mind, we report herein a highly active palladium‐free Cu/carbazole‐based NHC catalyst system allowing homocoupling and heterocoupling of alkynes at ambient temperature. Using a combination of Cu/NHC‐catalyzed homocoupling/deprotection/heterocoupling sequence tetraynes have also been synthesized in good yields. The Cu/NHC catalyst system was also able to catalyze a palladium‐free Sonogashira reaction of synthetically useful nucleosides.

Novel synthetic strategy towards subphthalocyanine-functionalized acetylenic scaffolds via various dibromo-enynes.

Boron subphthalocyanine (SubPc) is a strong chromophore with interesting applications in the field of functional materials and can be synthetically modified in both the peripheral and axial positions, allowing tuning of optical and redox properties. Herein we present novel acetylenic scaffolds where SubPc units are bridged via acetylenic moieties at the boron atoms. Specifically, we show that dibromo-functionalized enyne and enediyne units (vinylic dibromides) can be attached to one or two SubPc boron atoms using an AlCl3-mediated alkynylation protocol of trimethylsilyl-protected alkynes, and such compounds can conveniently be employed for further Sonogashira or Glaser-Hay coupling reactions. Thereby, new tetraethynylethene (TEE) - SubPc scaffolds were obtained. The degree of communication between two SubPc units incorporated in dimeric scaffolds was investigated by cyclic voltammetry. When bridged by one TEE unit, the oxidations of the SubPc units occurred sequentially, while the two SubPc units behaved as independent redox centers when separated by two TEE units.

Silver-catalyzed acylative annulation of N-propargylated indoles with α-keto acids: access to acylated pyrrolo[1,2-a]indoles.

This study describes the first carbon centered radical cyclization of N-propargyl indoles involving decarboxylation of α-keto acids in the presence of an Ag-catalyst. The present cascade carbon-carbon bond formation reactions provide C-acylated pyrrolo[1,2-a]indoles in good yields (25 examples). The amount of oxidant has a profound effect on the reaction to convert either the di-acylated products using 8 equiv. of K2S2O8 or mono-acylated products using 2 equiv. of K2S2O8. N-Propargyl pyrroles are also found to be suitable for delivering the corresponding 3H-pyrrolizines.

Gold-catalyzed dual C-C bond cleavage of biphenylenes bearing a pendant alkyne at ambient temperature.

We report a catalytic skeletal rearrangement of biphenylenes with a pendant alkyne moiety at room temperature by a cationic gold catalyst, which involves the cleavage of two bonds: the C-C bond of biphenylene and the C(sp)-C(sp2 or sp3) bond. Experimental and theoretical studies revealed that the reaction mechanism included π-activation of the alkyne, ring expansion and 1,2-carbon shift.

An Expeditious Route for the Synthesis of Oxazepine Triazolo-β-Lactams through Intramolecular Metal-Free [3+2] Azide–Alkyne Cycloaddition

A copper-free intramolecular azide–alkyne cycloaddition reaction of 4-hydroxymethyl-β-lactam with sodium azide has been described. The present approach involves the incorporation of an alkyne moiety through O-alkynylation of 3-hydroxy β-lactam with various propargylic halides. The generality of the method has been demonstrated by treating the corresponding tosylates or mesylates of the hydroxymethyl functionality of a variety of β-lactam-tethered terminal and internal alkynes with sodium azide in a one-pot three-step reaction to furnish novel oxazepane-β-lactam fused triazole scaffolds of diverse interest in good yield.

Expedient synthesis of conjugated triynes via alkyne metathesis.

The first synthesis of conjugated triynes by molybdenum-catalysed alkyne metathesis is reported. Strategic to the success of this approach is the utilization of sterically-hindered diynes that allowed for the site-selective alkyne metathesis to produce the desired conjugated triyne products. The steric hindrance of the alkyne moiety was found to be crucial in preventing the formation of diyne byproducts. This novel synthetic strategy was amenable to self- and cross-metathesis providing straightforward access to the corresponding symmetrical and dissymmetrical triynes with high selectivity.

Target identification of a macrocyclic hexaoxazole G-quadruplex ligand using post-target-binding visualization.

Macrocyclic hexaoxazoles (6OTDs) are G-quadruplex (G4) ligands, and some derivatives, such as L2H2-6OTD (1a) bearing two aminobutyl side chains, show cytotoxicity towards cancer cells. To identify the cellular target of 1a, we employed a post-target-binding strategy utilizing click reaction (Huisgen cyclization) between the azide-conjugated ligand L2H2-6OTD-Az (1b) and the cell-permeable dye CO-1 bearing a strained alkyne moiety and the BODIPY fluorophore under Cu-free conditions. We confirmed that introduction of the small azide group did not alter the physical or biological properties, including anti-cancer activity, of 1a, and we also demonstrated bias-free localization of CO-1. The post-binding visualization strategy suggested that L2H2-6OTD (1a) colocalized with RNA G4 in living cells.