Thioamides in Nature: In Search of Secondary Metabolites in Anaerobic Microorganisms

Abstract

The importance of S: Thioamide peptides and sulfur-containing natural compounds are highlighted, with an emphasis on closthioamide (X=S). The properties and functions of thioamide-containing peptides and their biological activities are discussed. Thioamides are formed by replacing the carbonyl oxygen in an amide bond with sulfur (HNCS), and have been considered as an isosteric replacement.1 Biophysicists and medicinal chemists employ thioamide substitutions in the backbone of peptides and other amide-containing compounds. Several examples have been reported that improve stability in proteolytic degradation and improve the ADME properties of amide-containing compounds.2, 3 In several cases, the thioamide-modified peptides exhibited improved (bio)activity as well. In addition, thioamides are used in biophysical measurements to investigate the structure, stability and conformations of peptides. This is due to the fact that the larger sulfur atom and, thus, the elongated CS bond (1.67 Å) induce conformational changes and a higher rotation barrier around the CN bond.2 Apart from this, the propensity for hydrogen bond formation is altered in thioamides, as NH is a better H-donor but CS is a weaker H-acceptor than in amides.3 Finally, the thioamide CS bond has an UV absorption maximum at 265(±5) nm and an IR stretch at 1120(±20) cm−1, compared to the 220(±5) nm and 1660(±20) cm−1, respectively, of a CO amide bond. Moreover, the 13C NMR shifts differ by ∼30 ppm. Practical and simple routes have been developed for the synthesis of thioamides from amides by using phosphorus pentasulfide and Lawesson's reagent.4 Remarkably, until recently, only four thioamide compounds 1 were known out of over 170 000 natural products. Though 2 is believed to be an artefact in isolation,5 cycasthioamide (1)6 is of plant origin. Only thioviridamide (3)7 and apo-methanobactin (4, structure revised),8, 9 are of bacterial origin, isolated from Streptomyces olivoviridis and Methylosinus trichosporium, respectively. Very little is known about secondary metabolites from anaerobic bacteria.10, 11 The genus Clostridium, which is known for its pathogenic strains C. botulinum and C. tetani, contains about 150 metabolically diverse anaerobic species wherever organic matter—including soils, aquatic sediments and the intestinal tracts of animals and humans—is present. Clostridium cellulolyticum is an obligate, anaerobic, Gram-positive bacterium and it represents an important industrial strain due to its ability to degrade crystalline cellulose. However, until recently, no C. cellulolyticum secondary metabolites were known from standard laboratory cultures. Upon genomic analysis, Hertweck and colleagues expected that some of these genes could be responsible for secondary metabolites, but that they were in a latent phase in standard cultures. Therefore C. cellulolyticum was grown to activate latent genes by employing external triggers, such as chemical supplements and pH and temperature stress; however, initially without success.12 As C. cellulolyticum has as its natural habitat decayed grass or soil compost, natural conditions were induced by adding aqueous soil extracts to the cultures. From these cultures a new all-thioamide compound, named closthioamide (5), 1has been isolated. Its structure was elucidated by HRMS and NMR spectroscopy and found to contain six thioamides.12 This seemingly simple, symmetric, twin, drug-like hexathioamide, which is assembled mainly from β-alanine and 4-hydroxy benzoic acid analogues, showed good antibacterial activity (MIC=0.58 μM) in methillicin-resistant Staphylococcus aureus (MRSA) strains as well as vancomycin-resistant Enterococcus faecalis (VRE) strains. Its antibacterial activity surpassed that of the current standard drug against MRSA and VRE, ciprofloxacin, thus attracting more attention. Closthioamide showed moderate cytotoxicity (CC50=10.23 μM, HeLa cells), as well as antiproliferative activity (GI50=9.22 μM, HUVEC cells and GI50=2.16 μM K-562 cells). The preliminary data encourage further testing on its mode of antibacterial activity. The Hertweck group confirmed that the thioamide bonds were the key to the molecule's antibacterial activity by synthesising the amide analogue 6. More in-depth studies are needed in this area to elucidate further structure–activity relationships. In this context, future genomic studies have to show whether the biosynthetic repertoire of anaerobes, with regard to secondary metabolites, is limited to a few structurally simple molecules or whether they are also able to synthesise more complex metabolites. This is important because molecular oxygen, a substrate for P450 monooxygenases and thus a precursor to many structural complexities in secondary metabolites, would have to be substituted through alternative biosynthetic pathways. Sulfur is an interesting constituent of various secondary metabolites. Besides the thioamide peptides, these comprise lantibiotics (thioether),13 and heterocyclic thiazole peptides (microcin B17).14 In this context, the question of the mechanisms of introduction of the sulfur atoms into those molecules arises. Over 80 natural thiazole-containing antibiotic compounds, called thiopeptides15 (e.g., thiostreptone), have been isolated, and their biosynthesis is well understood. Well-established thiazole heterocycles originate from cyclodehydration of the amide bond with the Cys (X-Cys) side-chain thiol followed by oxidation. This cyclodehydration of X-Cys peptide linkages is not restricted to peptides synthesised by nonribosomal peptide synthetase (NRPS) assembly, ribosomally synthesised peptides can undergo the same type of modification. In contrast, for the thioamide-containing, copper-binding siderophore apo-methanobactin (4), it was postulated that sulfuration of the amide bond adjacent to Ser (of the precursors of the oxazolone rings A and B) occurs as the first step in the biosynthesis of the 11-membered linear peptide.9 Sulfuration was suggested to occur by the attack of enzyme-bound Cys or CoA-sulfur, followed by the action of a lyase to incorporate the thioamide. Sulfur-containing enzymes are ubiquitous in Nature; however, none that performs the sulfuration of amides or functions as a thiosynthase or sulfur transferase has been described. In the respiration of anaerobic microorganisms, including C. cellulolyticum, hydrogen sulfide (H2S) is used for redox processes in the oxygen-free environment, and atomic sulfur and other sulfites are synthesised. In a putative biosynthesis of closthioamide, the introduction of sulfur could occur in an early (before amide formation) or late (amide to thioamide replacement) biosynthetic step. The first model would involve a differentiation in the leaving group: if a CoA-enzyme-activated thioacyl derivative undergoes ligation, carbonyl oxygen would be the leaving group, or O-thioacyl to S-thioacyl rearrangement occurs before the ligation. If sulfur replacement occurs in the carboxylic acid itself, there would be a problem in chemical differentiation between the two possible thiocarboxylic acids. In this context, it would have been interesting to see whether oxo-derivatives of closthioamide could also be isolated from C. cellulolyticum. In the latter model, either enzyme-bound sulfur or activated H2S is inserted into the amide-bond-forming thioamides. The precursor peptide might then be synthesised from 4-hydroxy benzoic acid and β-alanines by NRPSs.16 No concrete information is available on the origins of 1,3-diamino propane, the related natural diamine norspermidine has been found in the bacterial siderophore vibriobactin biosynthesis of Vibrio cholerae.17 Although the biochemical function of closthioamide in C. cellulolyticum is yet be explored, due to its high symmetry and six sulfurs, one may predict a soft ligand nature and a putative function as a siderophore or siderophore-like compound. Medicinal chemists commonly use thioamide as an isosteric substitution for amides, one of various backbone modifications in the search for more potent and stable compounds than the parent physiologically active peptides and small molecules. The replacement of proteolytically sensitive amide bonds has been an especially successful strategy in the design of novel inhibitors. Second-line antituberculosis drugs, 2-ethyl/2-propyl 4-thiocarbamoylpyridine (ethionamide/prothionamide), are thioamide-containing drugs and have been in clinical application for decades. However, their amide analogues, such as 2-ethyl/2-propyl isonicotinamide have no antituberculosis activity, and are only isolated as metabolites. Similarly thiourea-based drugs, such as thioacetazone, ethyl/propyl thiouracil and noxytiolin, have no urea (amide) analogues in medicinal applications, thus showing the importance of thioamides in their bioactivity. The discovery of closthioamide (5) raises the interest of the natural product community in searching for other natural anaerobic secondary metabolites, which are dormant under standard culture conditions. The biosynthetic pathways in anaerobes, whether further secondary metabolites can be identified and to what extent they are limited in structural complexity also remain to be explored. The authors acknowledge support from the Cluster of Excellence “Unifying Concepts in Catalysis” coordinated by the Technische Universität Berlin, and S.B. acknowledges the Alexander-von-Humboldt-Stiftung for a postdoctoral fellowship.