A terpene nucleoside from M. tuberculosis induces lysosomal lipid storage in foamy macrophages.

Induction of lipid-laden foamy macrophages is a cellular hallmark of tuberculosis (TB) disease, which involves the transformation of infected phagolysosomes from a site of killing into a nutrient-rich replicative niche. Here, we show that a terpenyl nucleoside shed from Mycobacterium tuberculosis, 1-tuberculosinyladenosine (1-TbAd), caused lysosomal maturation arrest and autophagy blockade, leading to lipid storage in M1 macrophages. Pure 1-TbAd, or infection with terpenyl nucleoside-producing M. tuberculosis, caused intralysosomal and peribacillary lipid storage patterns that matched both the molecules and subcellular locations known in foamy macrophages. Lipidomics showed that 1-TbAd induced storage of triacylglycerides and cholesterylesters and that 1-TbAd increased M. tuberculosis growth under conditions of restricted lipid access in macrophages. Furthermore, lipidomics identified 1-TbAd-induced lipid substrates that define Gaucher's disease, Wolman's disease, and other inborn lysosomal storage diseases. These data identify genetic and molecular causes of M. tuberculosis-induced lysosomal failure, leading to successful testing of an agonist of TRPML1 calcium channels that reverses lipid storage in cells. These data establish the host-directed cellular functions of an orphan effector molecule that promotes survival in macrophages, providing both an upstream cause and detailed picture of lysosome failure in foamy macrophages.

Design and Synthesis of Visible-Light-Responsive Azobenzene Building Blocks for Chemical Biology.

Tetra-ortho-fluoro-azobenzenes are a class of photoswitches useful for the construction of visible-light-controlled molecular systems. They can be used to achieve spatio-temporal control over the properties of a chosen bioactive molecule. However, the introduction of different substituents to the tetra-fluoro-azobenzene core can significantly affect the photochemical properties of the switch and compromise biocompatibility. Herein, we explored the effect of useful substituents, such as functionalization points, attachment handles, and water-solubilizing groups, on the photochemical properties of this photochromic system. In general, all the tested fluorinated azobenzenes exhibited favorable photochemical properties, such as high photostationary state distribution and long half-lives, both in organic solvents and in water. One of the azobenzene building blocks was functionalized with a trehalose group to enable the uptake of the photoswitch into mycobacteria. Following metabolic uptake and incorporation of the trehalose-based azobenzene in the mycobacterial cell wall, we demonstrated photoswitching of the azobenzene in the isolated total lipid extract.

Pd-catalyzed sp-sp3 cross-coupling of benzyl bromides using lithium acetylides.

Organolithium-based cross-coupling reactions have emerged as an indispensable method to construct C-C bonds. These transformations have proven particularly useful for the direct and fast coupling of various organolithium reagents (sp, sp2, and sp3) with aromatic (pseudo) halides (sp2). Here we present an efficient method for the cross-coupling of benzyl bromides (sp3) with lithium acetylides (sp). The reaction proceeds within 10 min at room temperature and can be performed in the presence of organolithium-sensitive functional groups such as esters, nitriles, amides and boronic esters. The potential application of the methodology is demonstrated in the preparation of key intermediates used in pharmaceuticals, chemical biology and natural products.

Chemical Synthesis of Cell Wall Constituents of Mycobacterium tuberculosis.

The pathogen Mycobacterium tuberculosis (Mtb), causing tuberculosis disease, features an extraordinary thick cell envelope, rich in Mtb-specific lipids, glycolipids, and glycans. These cell wall components are often directly involved in host-pathogen interaction and recognition, intracellular survival, and virulence. For decades, these mycobacterial natural products have been of great interest for immunology and synthetic chemistry alike, due to their complex molecular structure and the biological functions arising from it. The synthesis of many of these constituents has been achieved and aided the elucidation of their function by utilizing the synthetic material to study Mtb immunology. This review summarizes the synthetic efforts of a quarter century of total synthesis and highlights how the synthesis layed the foundation for immunological studies as well as drove the field of organic synthesis and catalysis to efficiently access these complex natural products.

Chiral Amplification of Phosphoramidates of Amines and Amino Acids in Water.

The origin of biomolecular homochirality continues to be one of the most fascinating aspects of prebiotic chemistry. Various amplification strategies for chiral compounds to enhance a small chiral preference have been reported, but none of these involves phosphorylation, one of nature's essential chemical reactions. Here we present a simple and robust concept of phosphorylation-based chiral amplification of amines and amino acids in water. By exploiting the difference in solubility of a racemic phosphoramidate and its enantiopure form, we achieved enantioenrichment in solution. Starting with near racemic, phenylethylamine-based phosphoramidates, ee's of up to 95 % are reached in a single amplification step. Particularly noteworthy is the enantioenrichment of phosphorylated amino acids and their derivatives, which might point to a potential role of phosphorus en-route to prebiotic homochirality.

Heterologous Production of 1-Tuberculosinyladenosine in Mycobacterium kansasii Models Pathoevolution towards the Transcellular Lifestyle of Mycobacterium tuberculosis.

Mycobacterium kansasii is an environmental nontuberculous mycobacterium that causes opportunistic tuberculosis-like disease. It is one of the most closely related species to the Mycobacterium tuberculosis complex. Using M. kansasii as a proxy for the M. kansasii-M. tuberculosis common ancestor, we asked whether introducing the M. tuberculosis-specific gene pair Rv3377c-Rv3378c into M. kansasii affects the course of experimental infection. Expression of these genes resulted in the production of an adenosine-linked lipid species, known as 1-tuberculosinyladenosine (1-TbAd), but did not alter growth in vitro under standard conditions. Production of 1-TbAd enhanced growth of M. kansasii under acidic conditions through a bacterial cell-intrinsic mechanism independent of controlling pH in the bulk extracellular and intracellular spaces. Production of 1-TbAd led to greater burden of M. kansasii in the lungs of C57BL/6 mice during the first 24 h after infection, and ex vivo infections of alveolar macrophages recapitulated this phenotype within the same time frame. However, in long-term infections, production of 1-TbAd resulted in impaired bacterial survival in both C57BL/6 mice and Ccr2-/- mice. We have demonstrated that M. kansasii is a valid surrogate of M. tuberculosis to study virulence factors acquired by the latter organism, yet shown the challenge inherent to studying the complex evolution of mycobacterial pathogenicity with isolated gene complementation.IMPORTANCE This work sheds light on the role of the lipid 1-tuberculosinyladenosine in the evolution of an environmental ancestor to M. tuberculosis On a larger scale, it reinforces the importance of horizontal gene transfer in bacterial evolution and examines novel models and methods to provide a better understanding of the subtle effects of individual M. tuberculosis-specific virulence factors in infection settings that are relevant to the pathogen.

Total Synthesis of a Mycolic Acid from Mycobacterium tuberculosis.

In Mycobacterium tuberculosis, mycolic acids and their glycerol, glucose, and trehalose esters ("cord factor") form the main part of the mycomembrane. Despite their first isolation almost a century ago, full stereochemical evaluation is lacking, as is a scalable synthesis required for accurate immunological, including vaccination, studies. Herein, we report an efficient, convergent, gram-scale synthesis of four stereo-isomers of a mycolic acid and its glucose ester. Binding to the antigen presenting protein CD1b and T cell activation studies are used to confirm the antigenicity of the synthetic material. The absolute stereochemistry of the syn-methoxy methyl moiety in natural material is evaluated by comparing its optical rotation with that of synthetic material.

Mycobacterium tuberculosis releases an antacid that remodels phagosomes.

Mycobacterium tuberculosis (Mtb) is the world's most deadly pathogen. Unlike less virulent mycobacteria, Mtb produces 1-tuberculosinyladenosine (1-TbAd), an unusual terpene nucleoside of unknown function. In the present study 1-TbAd has been shown to be a naturally evolved phagolysosome disruptor. 1-TbAd is highly prevalent among patient-derived Mtb strains, where it is among the most abundant lipids produced. Synthesis of TbAd analogs and their testing in cells demonstrate that their biological action is dependent on lipid linkage to the 1-position of adenosine, which creates a strong conjugate base. Furthermore, C20 lipid moieties confer passage through membranes. 1-TbAd selectively accumulates in acidic compartments, where it neutralizes the pH and swells lysosomes, obliterating their multilamellar structure. During macrophage infection, a 1-TbAd biosynthesis gene (Rv3378c) confers marked phagosomal swelling and intraphagosomal inclusions, demonstrating an essential role in regulating the Mtb cellular microenvironment. Although macrophages kill intracellular bacteria through phagosome acidification, Mtb coats itself abundantly with antacid.

Total Synthesis of an Immunogenic Trehalose Phospholipid from Salmonella Typhi and Elucidation of Its sn-Regiochemistry by Mass Spectrometry.

Diphosphatidyltrehalose (diPT) is an immunogenic glycolipid, recently isolated from Salmonella Typhi. Despite rigorous structure elucidation, the sn-position of the acyl chains on the glycerol backbone had not been unequivocally established. A stereoselective synthesis of diPT and its regioisomer is reported herein. Using a hybrid MS3 approach combining collisional dissociation and ultraviolet photodissociation mass spectrometry for analysis of the regioisomers and natural diPT, the regiochemistry of the acyl chains of this abundant immunostimulatory glycolipid was established.

Discovery of Salmonella trehalose phospholipids reveals functional convergence with mycobacteria.

Salmonella species are among the world's most prevalent pathogens. Because the cell wall interfaces with the host, we designed a lipidomics approach to reveal pathogen-specific cell wall compounds. Among the molecules differentially expressed between Salmonella Paratyphi and S. Typhi, we focused on lipids that are enriched in S. Typhi, because it causes typhoid fever. We discovered a previously unknown family of trehalose phospholipids, 6,6'-diphosphatidyltrehalose (diPT) and 6-phosphatidyltrehalose (PT). Cardiolipin synthase B (ClsB) is essential for PT and diPT but not for cardiolipin biosynthesis. Chemotyping outperformed clsB homology analysis in evaluating synthesis of diPT. DiPT is restricted to a subset of Gram-negative bacteria: large amounts are produced by S. Typhi, lower amounts by other pathogens, and variable amounts by Escherichia coli strains. DiPT activates Mincle, a macrophage activating receptor that also recognizes mycobacterial cord factor (6,6'-trehalose dimycolate). Thus, Gram-negative bacteria show convergent function with mycobacteria. Overall, we discovered a previously unknown immunostimulant that is selectively expressed among medically important bacterial species.

Stereoselective Synthesis of 1-Tuberculosinyl Adenosine; a Virulence Factor of Mycobacterium tuberculosis.

Despite its status as one of the world's most prevalent and deadly bacterial pathogens, Mycobacterium tuberculosis (Mtb) infection is not routinely diagnosed by rapid and highly reliable tests. A program to discover Mtb-specific biomarkers recently identified two natural compounds, 1-tuberculosinyl adenosine (1-TbAd) and N(6)-tuberculosinyl adenosine (N(6)-TbAd). Based on their association with virulence, the lack of similar compounds in nature, the presence of multiple stereocenters, and the need for abundant products to develop diagnostic tests, synthesis of these compounds was considered to be of high value but challenging. Here, a multigram-scale stereoselective synthesis of 1-TbAd and N(6)-TbAd is described. As a key-step, a chiral auxiliary-mediated Diels-Alder cycloaddition was developed, introducing the three stereocenters with a high exo endo ratio (10:1) and excellent enantioselectivity (>98% ee). This constitutes the first entry into the stereoselective synthesis of diterpenes with the halimane skeleton. Computational studies explain the observed stereochemical outcome.

Palladium-Catalyzed, tert-Butyllithium-Mediated Dimerization of Aryl Halides and Its Application in the Atropselective Total Synthesis of Mastigophorene†A.

A palladium-catalyzed direct synthesis of symmetric biaryl compounds from aryl halides in the presence of tBuLi is described. In situ lithium-halogen exchange generates the corresponding aryl lithium reagent, which undergoes a homocoupling reaction with a second molecule of the aryl halide in the presence of the palladium catalyst (1 mol %). The reaction takes place at room temperature, is fast (1 h), and affords the corresponding biaryl compounds in good to excellent yields. The application of the method is demonstrated in an efficient asymmetric total synthesis of mastigophorene A. The chiral biaryl axis is constructed with an atropselectivity of 9:1 owing to catalyst-induced remote point-to-axial chirality transfer.

In vivo biosynthesis of terpene nucleosides provides unique chemical markers of Mycobacterium tuberculosis infection.

Although small molecules shed from pathogens are widely used to diagnose infection, such tests have not been widely implemented for tuberculosis. Here we show that the recently identified compound, 1-tuberculosinyladenosine (1-TbAd), accumulates to comprise >1% of all Mycobacterium tuberculosis lipid. In vitro and in vivo, two isomers of TbAd were detected that might serve as infection markers. Using mass spectrometry and nuclear magnetic resonance, we established the structure of the previously unknown molecule, N(6)-tuberculosinyladenosine (N(6)-TbAd). Its biosynthesis involves enzymatic production of 1-TbAd by Rv3378c followed by conversion to N(6)-TbAd via the Dimroth rearrangement. Intact biosynthetic genes are observed only within M. tuberculosis complex bacteria, and TbAd was not detected among other medically important pathogens, environmental bacteria, and vaccine strains. With no substantially similar known molecules in nature, the discovery and in vivo detection of two abundant terpene nucleosides support their development as specific diagnostic markers of tuberculosis.

Enantioselective palladium catalyzed conjugate additions of ortho-substituted arylboronic acids to ß,ß-disubstituted cyclic enones: total synthesis of herbertenediol, enokipodin A and enokipodin B.

The palladium-catalyzed conjugate addition (Michael addition) of ortho-substituted arylboronic acids to ß,ß-disubstituted cyclic enones, in particular 3-methyl cyclopent-2-enone and 3-methyl cyclohex-2-enone, is reported. With an achiral bipyridine-based palladium catalyst, good yields are obtained with a variety of ortho-substituted arylboronic acids. In the asymmetric version, good to very high enantiomeric excesses (up to 99% ee) are obtained, though the yields are moderate. The decreased yields are attributed to significant protodeboronation of the arylboronic acid. The developed methodology allows the efficient enantioselective synthesis of the very crowded, biologically active, sesquiterpenes herbertenediol, enokipodin A, and enokipodin B.

Asymmetric total synthesis of a putative sex pheromone component from the parasitoid wasp Trichogramma turkestanica.

Virgin females of the parasitoid wasp Trichogramma turkestanica produce minute amounts of a sex pheromone, the identity of which has not been fully established. The enantioselective synthesis of a putative component of this pheromone, (6S,8S,10S)-4,6,8,10-tetramethyltrideca-2E,4E-dien-1-ol (2), is reported as a contribution to this identification. Catalytic asymmetric conjugate addition of methylmagnesium bromide and stereoselective Horner-Wadsworth-Emmons olefinations are used as the key steps, and 2 was obtained in 16 steps with an overall yield of 4.4%.

Molecular profiling of Mycobacterium tuberculosis identifies tuberculosinyl nucleoside products of the virulence-associated enzyme Rv3378c.

To identify lipids with roles in tuberculosis disease, we systematically compared the lipid content of virulent Mycobacterium tuberculosis with the attenuated vaccine strain Mycobacterium bovis bacillus Calmette-Guérin. Comparative lipidomics analysis identified more than 1,000 molecular differences, including a previously unknown, Mycobacterium tuberculosis-specific lipid that is composed of a diterpene unit linked to adenosine. We established the complete structure of the natural product as 1-tuberculosinyladenosine (1-TbAd) using mass spectrometry and NMR spectroscopy. A screen for 1-TbAd mutants, complementation studies, and gene transfer identified Rv3378c as necessary for 1-TbAd biosynthesis. Whereas Rv3378c was previously thought to function as a phosphatase, these studies establish its role as a tuberculosinyl transferase and suggest a revised biosynthetic pathway for the sequential action of Rv3377c-Rv3378c. In agreement with this model, recombinant Rv3378c protein produced 1-TbAd, and its crystal structure revealed a cis-prenyl transferase fold with hydrophobic residues for isoprenoid binding and a second binding pocket suitable for the nucleoside substrate. The dual-substrate pocket distinguishes Rv3378c from classical cis-prenyl transferases, providing a unique model for the prenylation of diverse metabolites. Terpene nucleosides are rare in nature, and 1-TbAd is known only in Mycobacterium tuberculosis. Thus, this intersection of nucleoside and terpene pathways likely arose late in the evolution of the Mycobacterium tuberculosis complex; 1-TbAd serves as an abundant chemical marker of Mycobacterium tuberculosis, and the extracellular export of this amphipathic molecule likely accounts for the known virulence-promoting effects of the Rv3378c enzyme.

A protecting group-free synthesis of the Colorado potato beetle pheromone.

A novel synthesis of the aggregation pheromone of the Colorado potato beetle, Leptinotarsa decemlineata, has been developed based on a Sharpless asymmetric epoxidation in combination with a chemoselective alcohol oxidation using catalytic [(neocuproine)PdOAc]2OTf2. Employing this approach, the pheromone was synthesized in 3 steps, 80% yield and 86% ee from geraniol.

Synthesis and analysis of the all-(S) side chain of phosphomycoketides: a test of NMR predictions for saturated oligoisoprenoid stereoisomers.

(4S,8S,12S,16S,20S)-Pentamethylheptacosan-1-ol has been synthesized and analyzed by resolution-enhanced NMR spectroscopy with the aid of a recent set predicted spectra of all its stereoisomers. The configuration was confirmed, but isomer purity of the sample (~70%) was lower than expected. A truncated analogue, (2S,6S,10S,14S)-2,6,10,14-tetramethylhenicosan-1-ol TBDPS ether, was prepared from a late stage synthetic intermediate. Analysis of its spectra confirmed the configuration and showed that the sample was isomerically pure. The results suggest that a late-stage epimerization, not a failure of an asymmetric synthesis step, caused the formation of minor stereoisomers in the sample of pentamethylheptacosan-1-ol. The study shows the value of the predicted set of oligoisoprenoid spectra and further extends the predictive model to a new subclass of compounds.