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.TbAd | terpenyl transferase W ith a mortality rate exceeding 1.5 million deaths annually, Mycobacterium tuberculosis remains one of the world's most important pathogens (1). M. tuberculosis succeeds as a pathogen because of productive infection of the endosomal network of phagocytes. Its residence within the phagosome protects it from immune responses during its decades long infection cycle. However, intracellular survival depends on active inhibition of pH-dependent killing mechanisms, which occurs for M. tuberculosis but not species with low disease-causing potential (2). Intracellular survival is also enhanced by an unusually hydrophobic and multilayered protective cell envelope. Despite study of this pathogen for more than a century, the spectrum of natural lipids within M. tuberculosis membranes is not yet fully defined. For example, the products of many genes annotated as lipid synthases remain unknown (3), and mass spectrometry detects hundreds of ions that do not correspond to known lipids in the MycoMass and LipidDB databases (4, 5).To broadly compare the lipid profiles of virulent and avirulent mycobacteria, we took advantage of a recently validated metabolomics platform (4). This high performance liquid chromatography-mass spectrometry (HPLC-MS)...

ycobacterium tuberculosis kills more humans than any other pathogen 1 . Whereas most bacterial pathogens cause acute disease, Mtb usually undergoes a years-long infection cycle. Mtb persists in humans in part through parasitism of macrophage phagosomes. Survival in this intracellular niche is accomplished by slowing phagosomal maturation and reducing intracellular killing mechanisms 2-4 , while offering partial cloaking from immune cells and access to lipids and other host nutrients 5,6 . As Mtb interactions with the host play out over years and at diverse anatomical sites, pinpointing specific events that determine tuberculosis (TB) disease outcome is challenging. However, a successful approach has been the comparative profiling of mycobacteria of varying virulence to discover factors selectively present in highly virulent species. Mycobacterium species naturally differ in their potential to infect, persist and cause TB, and transmit among hosts. With an estimated 1.7 billion infections worldwide 1 , only Mtb has broadly colonized the human species, and humans represent its only natural host. These observations highlight the need to identify factors selectively expressed in Mtb but not in other mycobacterial species.Comparative genomics and transcriptomics of Mtb and Bacille Calmette-Guèrin (BCG) have isolated factors selectively present in Mtb, such as the ESX-1 transporter 7 . Whereas genetic techniques are widely used, comparative chemical biology screens are uncommon in mycobacteria. An HPLC-mass spectrometry (MS)-based lipidomics platform was developed for analysis of all chloroform/methanol-extractable mycobacterial lipids 8,9 . Comparative lipidomics of Mtb and BCG identified a previously unknown, Mtb-specific lipid missed by genomics approaches: 1-tuberculosinyladenosine (1-TbAd, 1) 10 . Cyclization of geranylgeranyl pyrophosphate into tuberculosinyl pyrophosphate occurs via the enzyme, Rv3377c, and tuberculosinyl transferase (Rv3378c) generates 1-TbAd, which can chemically rearrange to N 6 -TbAd (2) [10][11][12] . So far 1-TbAd has been detected only in Mtb 12 , so its expression correlates with evolved virulence. However, 1-TbAd has been studied only in laboratoryadapted strains 12,13 , and the extent to which it is produced by patientderived Mtb strains remains unknown.Furthermore, 1-TbAd's function remains unknown. Transposon inactivation of Rv3377c or Rv3378c reduced Mtb uptake, phagosomal acidification and killing of Mtb in mouse macrophages 14 . Therefore, 1-TbAd might influence some aspects of these processes in host cells. However, any host receptor, receptor-independent mechanism or other target of 1-TbAd in host cells remains unknown. Commonly used bioinformatic predictors were not helpful for understanding 1-TbAd function, because it was not possible to identify orthologous biosynthetic genes or similar 1-linked purines in other species. Therefore, diverse candidate mechanisms

Summary 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 percent of all M. tuberculosis lipids. In vitro and in vivo, two isomers of TbAd were detected that might serve as infection markers. Using mass spectrometry and NMR, we established the structure of the previously unknown molecule, N6-tuberculosinyladenosine (N6-TbAd). Its biosynthesis involves enzymatic production of 1-TbAd by Rv3378c followed by conversion to N6-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.

Several 2,5-dialkylsubstituted 3-1,3,4-thiadiazolines have been prepared. On heating these afford unstable thiocarbonyl ylides, R2CSCR2, which are characterized by their tendency to yield episulfides on ring closure or cycloadducts on reaction with dipolarophiles such as dimethyl acetylenedicarboxylate or diethyl azodicarboxylate. The stereochemistry of these processes is in accord with predictions based on orbital symmetry considerations: conrotatory ring closure and retention of configuration during cycloaddition. The stereochemical behavior was established with cisand trans-2,5-diethyl-3-1,3,4-thiadiazoIines and cis-and ircms-2,5-di-fert-butyl-h3-l,3,4thiadiazolines. Syntheses of the thiadiazolines are accomplished by (a) condensation of a carbonyl compound, hydrazine, and hydrogen sulfide to afford a 1,3,4-thiadiazolidine that is subsequently dehydrogenated; (b) addition of hydrogen sulfide to an azine followed by dehydrogenation of the 1,3,4-thiadiazolidine; (c) reaction of hydrogen sulfide with the addition products of chlorine with azines. The activation parameters for decomposition of the thiadiazolines were determined. On the basis of steric considerations, thiocarbonyl ylides are concluded to be nonplanar. Factors affecting reactivity are discussed.The term "thiocarbonyl ylide"3 denotes the electrically neutral entity consisting of two trivalent carbon atoms bonded to a central sulfur atom. The simplest geometrical formulation is planar 1, which, from a pedagogical point of view, could be derived by the routes illustrated in eq 1. The components of 1, albeit greatly perturbed electronically and/or geometrically, are recognized in a diversity of compounds, including, for example, divinyl sulfide (2), thiophene (3), and 1,4dithiadiene (4).