Review – Lichen-Associated Bacteria as a Hot Spot of Chemodiversity: Focus on Uncialamycin, a Promising Compound for Future Medicinal Applications

Abstract

This review presents the state of knowledge on the medicinal potential of bacteria associated with lichens. In fact, besides the classical symbiotic partners (photobiont and mycobiont) forming the lichen thallus, associated bacteria have been recently described as a third partner. Various studies demonstrated the diversity of these communities with a predominance of Alphaproteobacteria. Bacterial groups more relevant for secondary metabolite synthesis have also been revealed. This article summarizes studies reporting the abilities of these communities to produce metabolites with relevant bioactivities. The biotechnological interest of these bacteria for drug discovery is highlighted regarding the production of compounds with therapeutic potential. Special focus is given to the synthesis of the most promising compound, uncialamycin, a potent enediyne isolated from a Streptomyces sp. associated with Cladonia uncialis.

Key words

lichens - bacterial communities - chemodiversity - drug discovery - bioactive compounds - uncialamycin

Introduction

Despite significant treatment advances, diseases such as cancer and microbial infections remain major public health issues due to the emergence of antibiotic resistance and the weak efficiency of current anticancer therapies. Therefore, biotechnological advances and the search of more specific and effective drugs are important challenges for pharmaceutical companies worldwide. The recent discovery of teixobactin from uncultivated soil bacteria highlights the unexplored microbial sources as treasure chests for the access to new interesting bioactive drugs [1]. Among underexplored sources, lichens are unique and are classically described as a symbiotic association between a photobiont (green alga and/or cyanobacterium) and a mycobiont. Most lichens are outstanding in their capacities to produce specific secondary metabolites that present biological activities, e.g., antioxidant, cytotoxic, and antimicrobial activities [2], [3], [4], [5], [6], [7], [8]. These organisms were also known to represent habitats for diverse lichenicolous fungi, with a high biosynthetic potential [9], [10]. Recent molecular approaches have also demonstrated the high diversity of lichen-associated bacterial communities that comprise millions of bacterial cells per gram of lichen thallus [11], [12], [13], [14], [15], [16], [17], [18], [19], [20], [21]. These associated organisms form stable and specific communities and thus represent as a whole a third partner of the lichens symbiosis [22]. They particularly colonize hydrophilic surfaces of lichens and are also incorporated within the extracellular fungal matrix [13]. These bacteria could either live as biofilm-like structures or as individual colonies [23]. These communities were characterized by culture-independent methods (e.g., pyrosequencing and FISH/CLSM) [12], [13], [14], [15], [17], [18], [20], [21], [24], [25], [26] and culture-dependent approaches [11], [13], [27], [28], [29], [30], [31], [32], [33], [34], [35], [36]. While proteobacteria usually dominate the bacterial community, members of other groups are present as well, in particular Actinobacteria, Bacteroidetes, Acidobacteria. In a recent review, we focused on the diversity of these communities, particularly in the point of view of the presence of biotechnologically interesting bacteria [37].

These species-specific diversity patterns of bacteria associated with lichens are influenced by various parameters such as extrinsic factors (e.g., exposure, substrate type, and location) and intrinsic factors (e.g., lichen species, age and part of the lichen thallus, and chemical composition of the lichen) [16], [17], [18], [23], [26], [38], [39], [40] ([Fig. 1]). Indeed some studies highlighted the prevalence of Acetobacteraceae (Alphaproteobacteria) associated with lichens, which is a proteobacterial family known to harbor nitrogen-fixing bacteria [13], [17], [18], [20], [25], [39]. Their presence may also be correlated to their ability to use carbon sources furnished by lichen mycobionts such as mannitol [20]. Moreover, it was reported prevalent secondary bioactive metabolites from lichens such as usnic acid, a abundant in some lichen species and with multiple biological activities (such as antibacterial, cytotoxic, and antifungal) [3], [4], [5], [6], only influenced the bacterial diversity but not its abundance. Accordingly, bacterial communities associated with lichens producing usnic acid were more specific and more resistant to this metabolite [23], [26]. All of these results suggest a closer relationship between the different partners.

Fig. 1 Model of relationships between lichen symbiosis partners. The model includes relevant functions of the colonizing bacteria, which are derived from -omic analysis as well as culture-dependent approaches (adapted from Grube et al., 2015) [22]. (Color figure available online only.)

The functional implications of associated bacteria in the lichen symbiosis remain to this day largely unexplained and unexplored. Nonetheless, some nonexclusive hypotheses have been proposed and/or reported. Globally, these hypotheses indicate that the symbiotic microbial communities in lichens might play an important role in (1) nutrient supply, especially nitrogen, phosphorus, and sulfur, (2) recycling nutrients, (3) resistance against biotic stress factors by the production of bioactive metabolites (e.g., uncialamycin, aminocoumarins, angucyclines, butenolides), (4) resistance against abiotic factors, (5) support of photosynthesis by the provision of vitamin B12, (6) fungal and algal growth support by the provision of hormones, (7) detoxification of metabolites, and (8) degradation of older parts of the lichen thallus [17], [19], [21], [22], [23], [24], [25], [41], [42] ([Fig. 1]). Even if bacteria or their metabolites could be present in a low amount in the holobiont, they could possibly play interesting ecological or biological roles. Further approaches to determine their localization inside the lichen thallus, the level of the activity of the produced metabolites, and the ability of the latter ones to dissipate into the thallus must be explored. A better understanding of the roles of small chemical compounds (called by Davies the “parvome” [43]) in cell-to-cell communication, particularly those produced by microbial organisms, will permit the development of rational approaches for the discovery of new drugs. In this context, lichen-associated bacteria could be interesting sources of bioactive natural molecules.

We will describe herein the isolation and the chemical characterization associated with the biological properties of compounds isolated from the cultivable bacteria [27], [28], [29], [30], [31], [32], [34], [44]. We will focus more precisely on uncialamycin, a compound isolated from a Streptomyces strain. Due to its potent DNA damage activity, this molecule with a reactive enediyne unit constitutes a valuable lead for pharmaceutical industries. As a result, intensive efforts have been implemented for the synthesis of this complex structure.

Biosynthetic Potential of Lichen-Associated Bacteria

The study of culturable lichen-associated bacterial communities led to the discovery of new bacterial species [27], [28], [29], [30], [31], [32], [34], [36], [45], [46]. Six new strains of Actinobacteria were thus isolated from unidentified lichens collected in Japan, e.g., Schumannella luteola sp. nov. [28], Leifsonia lichenia. sp. nov [27], Actinomycetospora iriomotensis sp. nov. [36], Nocardioides exalbidus sp. nov. [31], one from an unidentified lichen Actinoplanes sp. (MA7066 (ATCC 55 532) from Spain [46], and one strain Frondihabitans cladoniiphilus sp. nov. from Cladonia arbuscula [45]. Two new Streptomyces strains were isolated from lichens belonging to the Cladonia genus (Cladonia gracilis: strain L-44 [29] and C. uncialis: Streptomyces uncialis [30], [34], [44]) and four other Streptomyces sp. from lichens collected in Japan (strains RI104-LiC106 and RI104-LiB101) [32] or in Spain (Streptomyces cyaneofuscatus M-27 and S. carnosus M40) [47]. At least 14 novel culturable bacterial strains associated with lichens were thus identified and gave new opportunities to discover bioactive metabolites of interest. To date, the bacterial communities of only a few lichens have been studied as a source of original metabolites: Cladonia gracilis and Cladonia uncialis both collected in Canada and one unidentified lichen from Japan. Additionally, one recent study reports the presence of various interesting metabolites from S. cyaneofuscatus M-27 and S. carnosus M-40 isolated from unidentified lichens (e.g., antifungal compound maltophilin, antitumor daunomycin, or cosmomycin from the first strain or antibiotic and anti-inflammatory lodophorine B from the second one), but with the caveat that these identifications were only by HPLC [47] and production must eventually be confirmed by further chemical characterization. Notably, all reports concerning the production of interesting chemicals by lichen-associated bacteria deal with Actinobacteria, likely due to their known biotechnological potential, and even though it has been shown that Alphaproteobacteria are the predominant bacterial group on most lichens. Actinobacteria isolated from natural sources are widely recognized for their production of secondary metabolites with unusual structures and potent biological activities [48]. Among them, many known antimicrobials of major interest have been reported (e.g., erythromycin, streptomycin, and tetracycline). Moreover, relatively few interesting compounds were reported from Proteobacteria (including Alphaproteobacteria) in comparison with Actinobacteria [49]. As lichens host a significant number of Actinobacteria [35], [37], efforts have been made to isolate and study such bacteria from lichens, especially Streptomyces, as a promising resource for new lead compounds in drug development [37], [48]. This section provides the state of knowledge of the chemical potential of lichen-associated bacteria and demonstrates the real chemodiversity that these organisms could offer for medicinal applications. Secondary metabolites like angucyclin, the butenolide derivative JBIR-89, coumabiocins, uncialamycin, unciaphenol, and cladoniamides have been isolated from Streptomyces inhabiting lichens. Even though the ecological role of these compounds is still unclear, they have demonstrated remarkable biological activities (e.g., anticancer and antibacterial). Considering the complexity to identify them and their wide spectrum of biological activities, several research groups have achieved their total syntheses. Focus will be made on the total synthesis of uncialamycin and its analogues, as this molecule presents interesting cytotoxic activity associated with a specific mechanism of action.

Isolation and Bioactivity of Metabolites Produced by Culturable Bacteria

During their investigations, Motohashi and coworkers focused their attention on the isolation and chemical characterization of Streptomyces strains from a lichen collected at Risiri Island, Hokkaido prefecture, Japan [32]. By searching the homology of their 16S rRNA gene sequence with other bacteria, they identified strains RI104-LiC106 and RI104-LiB101 as new species of the genus Streptomyces. Partition using ethyl acetate was performed on an acetone crude extract of the mycelium produced during the fermentation (180 r. p. m., 27 °C, pH 7.2, 5 d). Chemical studies of the dried residues using reverse-phase chromatography led to the discovery of a new 1,1-dichlorocyclopropane-containing angucycline (1) from the culture broth of RI104-LiC106, and a new butenolide, designated as JBIR-89 (2), from RI104-LiB101 ([Fig. 2]). The isolation of angucycline 1 constituted the first report of a tetraphene containing a dichlorocyclopropane ring [32].

Fig. 2 Structures of angucycline 1 and the butenolide JBIR-89 (2).

Bioactivity studies on angucycline 1 showed antibacterial activity against Micrococcus luteus (diameter of inhibition zone: 11 mm, c = 25 µg on a 6-mm disk) and a lack of activity against Candida albicans and Escherichia coli. While angucycline 1 exhibited weak cytotoxic activity against HeLa (IC₅₀ = 36 µM) and ACC-MESO-1 (IC₅₀ = 52 µM) cells, the butenolide derivative (2) was not cytotoxic against either of the two cell lines [32].

Another Streptomycetaceae strain, Streptomyces L-4-4, sharing 98 % identity with the strain Streptomyces caeruleus based on 16S rRNA analysis, has been isolated from the surface of the lichen Cladonia gracilis from Mount Fromme in British Columbia using ISP4 (International Streptomyces Project 4) media plates supplemented with 50 µg/ml of cycloheximide and 20 µg/ml of nalidixic acid [29]. Studies of the butanol-soluble extract of fermented Streptomyces L-4-4 (250 r. p. m., 30 °C, pH 7.0, 7 d) highlighted an activity in hyphae formation inhibition (HFI) assays of prokaryotic whole cells [50], [51]. This analysis indicated the presence of protein kinase inhibitors in this extract. Further investigations of this extract led to the isolation of new aminocoumarins named coumabiocins A–F (3–8), along with two known compounds, novobiocin (9) and isonovobiocin (10). Coumabiocins A–E (3–7) contain three structural elements: a 3-amino-7-hydroxycoumarin core linked at the 3-amino group via a prenylated 4-hydroxybenzoic acid moiety and at the 7-position to an L-noviosyl sugar. Only coumabiocin F (8) lacks the sugar moiety ([Fig. 3]).

Fig. 3 Structures of coumabiocins A–F (3–8), novobiocin (9), and isonovobiocin (10).

The antimicrobial efficacy of novobiocin (9) had been confirmed in several clinical trials during the 1960s. After approval, it has been used as an antibiotic for the treatment of human infections [52], [53]. However, novobiocin is no longer used due to its toxicity. Significant inhibitory activities against Streptomyces 85E have been observed for coumabiocins A–E (3–7) (10–15 mm clear zone of inhibition at 20 µg/disk), while moderate activities were detected at lower concentrations (10 mm bald zone of inhibition at 2.5 µg/disk). Coumabiocin F (8), where the L-noviosyl sugar group is absent, was inactive, suggesting that the L-noviose group is essential for the activity in this class of molecules.

Studies of the bacterial communities of the lichen Cladonia uncialis (Pitt River, British Columbia) by J. Andersen and coworkers revealed a Streptomyces isolate putatively belonging to a new species named “S. uncialis” [30]. Based on its 16S rRNA sequences, this strain is related (98.4 %) to Streptomyces rubrogriseus. Laboratory cultures on ISP4 solid agar medium (30 °C, 14–21 d) followed by extractions with EtOAc and purification steps of the crude extract by flash C₁₈ reverse-phase chromatography and reverse-phase HPLC yielded uncialamycin (11), a new enediyne antibiotic. The structure of uncialamycin (11), similar to that of dynemicin A (12) isolated from Micromonospora chersina [54], combines a 10-membered enediyne with an anthraquinone substructure ([Fig. 4]).

Fig. 4 Structures of uncialamycin (11) and dynemicin A (12).

Structural similarities between uncialamycin (11) and dynemicin A (12) strongly suggest that they share the same biosynthetic pathway. Uncialamycin (11) could result from a degradation of a dynemicin-like precursor in which the C-5, C-6, and C-30 skeletal carbon atoms have been truncated. This metabolite showed in vitro antibacterial activities against gram-positive and gram-negative human pathogens, including Burkholderia cepacia (MIC = 0.001 µg/mL), a major cause of morbidity and mortality in patients with cystic fibrosis, methicillin-resistant Staphylococcus aureus (MIC = 0.0 000 064 µg/mL), and Escherichia coli (MIC = 0.002 µg/mL) [30]. Later, the synthesis of this compound and isomers confirmed the activities against B. cepacia (MIC = 0.0004 µg/mL), S. aureus (MIC = 0.0002 µg/mL), and allowed for the detection of further bioactivities on Staphylococcus epidermidis (MIC = 0.00 009 µg/mL), Streptococcus pneumoniae (MIC = 0.0004 µg/mL), and Enterococcus faecalis (MIC = 0.002 µg/mL) [55].

Enediyne compounds are remarkable active natural products, which own a unique and versatile core [56]. They are potent antitumor agents and have been studied extensively for use in the form of targeted antibody complexes [57], [58], [59]. To date, all natural enediynes exhibit cytotoxic effects as they act on duplex DNA and cause single- and double-stranded breaks. It is the result of the action of benzenoid diradicals formed by a Bergman rearrangement of the antibiotic molecule within the minor groove of the target DNA [60], [61], [62] ([Fig. 5]).

Fig. 5 Cycloaromatization of a (Z)-1,5-diyn-3-ene system.

Initial studies indicate that uncialamycin reacts with plasmid DNA leading to its degradation, and it is thus not surprising it presents potent activities against a broad panel of cancer cells, including Taxol-resistant ovarian cells (1A9/PTX10; IC₅₀ = 6 × 10⁻¹¹ M) and epothilone B-resistant cells (1A9/A8; IC₅₀ = 9 × 10⁻¹² M) [55]. Interestingly, an additional chemical study on this Streptomyces strain recently described the isolation of unciaphenol (13) by a supplementary purification using an LH-20 Sephadex column [44]. This compound was described as putative naturally occurring even if it corresponds to a Bergman cyclization product analogue of uncialamycin ([Fig. 6]). This compound was devoid of cytotoxicity, but exhibited an interesting in vitro anti-HIV activity against various strains resistant to antiretroviral compounds with an IC₅₀ range of 6.5 to 14.1 µM [44].

Fig. 6 Structures of unciaphenol and its putative obtention from uncialamycin (11).

Later, investigations of the EtOAc extracts of the culture of S. uncialis have resulted in the isolation of new alkaloids named cladoniamides A–G (14–20) ([Fig. 7]). These metabolites are derived of rearrangement and degradation of indolocarbazole precursors. The indolocarbazole alkaloids are a family of natural products isolated from marine invertebrates and cultures of diverse microorganisms [63].

Fig. 7 Structures of cladoniamides A–G (14–20).

During bioassays, only cladoniamide G (20) demonstrated cytotoxic activities on human breast cancer MCF-7 cells in vitro at 10 µg/mL. Indolocarbazole alkaloid derivatives such as cladoniamides are new aglycons that could be used to obtain new staurosporine/rebeccamycin (21/22) analogues ([Fig. 8]) by chemical and biotechnological approaches. Staurosporine and rebeccamycin have shown a high potential for the development of anticancer drugs [64], [65], [66], [67], [68], [69], as these two molecules are potent inhibitors of protein kinases and topoisomerase-1, respectively. Several analogues of staurosporine (21) and rebeccamycin (22) have entered clinical trials.

Fig. 8 Structures of staurosporine (21) and rebeccamycin (22).

Besides the study of various Streptomyces, one patent from Singh and coauthors reports the production of actinoplanic acids A and B ([Fig. 9]) from Actinoplanes sp. isolated from a Spanish lichen [46]. These 20-membered macrocyclic polycarboxylic acids exhibited potent and selective inhibition of farnesyl protein transferase with IC₅₀ of 230 nM and 50 nM respectively [46], [70], [71], and thus inhibited the farnesylation of the oncogene protein Ras, one interesting target for anticancer therapies [72].

Fig. 9 Structures of actinoplanic acids A (23) and B (24).

After the overview of the different metabolites isolated from lichen-associated bacteria, it appeared that these bacteria represent a very promising source of structurally diverse bioactive compounds. Reports of secondary metabolites from lichen-associated bacteria mention the use of various culture conditions of the target bacteria depending on their intrinsic characteristics. Parameters like temperature, pH, and media (solid or liquid) considerably impact the production of these metabolites. However, these culture conditions are very far from the in situ environment of the bacterial strains on the lichen, which are confronted with other biotic and abiotic factors. As a consequence, the metabolomic potential of these microorganisms are still poorly understood and, thus, underexploited. Despite these difficulties, the isolated metabolites belong to important classes of molecules (e.g., aminocoumarins, indolocarbazoles, alkaloids, enediynes). Uncialamycin is one of these examples as it presents a broad spectrum of antibacterial activities and interesting cytotoxic properties associated with an original mechanism of action. This atypical structure jointly with these impressive bioactivities has therefore generated interest for chemical synthesis.

Synthesis of Uncialamycin

Considering the small production yields of uncialamycin by S. uncialis and its high biological potential, different research groups have attempted the total synthesis of this metabolite. In the remainder of this section we will discuss two major synthetic strategies.

The first strategy, developed by Nicolaou and coworkers, is based on the use of the building blocks 25–27 and is centered around three key reactions for the formation of uncialamycin: i) an addition of an acetylide (26) to a pyridinium species followed by ii) an intramolecular addition of 26 to form the enediyne core and iii) a Hauser annulation with fragment 27 to form the anthraquinone moiety ([Fig. 10]) [73].

Fig. 10 Structures and retrosynthetic analysis of (26S)- and (26R)-uncialamycin (11a, 11b). DMB: 3,4-dimethoxybenzyl; TES: triethylsilyl; TMS: trimethylsilyl.

Fragment 25 was formed by starting with a two-step Friedländer quinoline synthesis [74]: condensation of 5-methoxyisatin (28) and methoxyenone (29) led to the ketocarboxylate 31 via intermediate 30; then 31 was reduced in situ to furnish the tricyclic lactone 32 with an 86 % overall yield ([Fig. 11]). Thereafter, a deprotection/protection step of the phenol moiety yielded compound 33 with a 50 % yield. After reduction of the lactone moiety and protection of the obtained lactol, the quinoline system 25 was achieved with an 86 % overall yield for the two steps and approximately a 1 : 1 mixture of diastereoisomers.

Fig. 11 Synthesis of quinoline 25.

The pyridine moiety of fragment 25 was activated by the formation of a pyridinium species, which was trapped by the acetylide derivative (26), generated from EtMgBr to afford the intermediate 34 with a 92 % yield ([Fig. 12]). After removal of the TES group, reduction of the lactol followed by a selective epoxidation and a monoacetylation yielded the hydroxyepoxide 35 (66 % overall yield for the three steps). After a protection/deprotection sequence leading to the formation of compound 36 (78 % overall yield for two steps), this latter one was submitted to an oxidation step of the hydroxyl group (87 % yield) and the deprotection of the phenol (87 % yield) to obtain the aldehyde 37. The cyclization, via an intramolecular acetylide addition, afforded the desired 10-membered ring enediyne 38 (61 % yield, 70 % ee). Compound 38 was then oxidized to a semiquinone system (80 % yield), and subsequent removal of the N-protective group furnished the iminoquinone 39 (74 % yield based on 70 % conversion). At last, Hauser annulation of 39 with nitrile 27 in the presence of LiHMDS (63 % yield) and desilylation (3HF×Et₃N, 92 % yield) gave (26 S)-uncialamycin (11a).

Fig. 12 Synthesis of (26S)-uncialamycin (11a). DDQ: 2,3-dichloro-5,6-dicyano-1,4-benzoquinone; DMP: Dess-Martin periodinane; KHMDS: potassium hexamethyldisilazide; mCPBA; meta-chloroperoxybenzoic acid.

Comparison of NMR data of isolated uncialamycin and the (26S)-isomer showed differences, which proved that the natural compound was not the 26S-isomer. To confirm these observations, Nicolaou and coworkers synthesized the 26R-epimer 11b. An oxidation/reduction sequence afforded the total inversion of the configuration at the C26 position (compound 41, 90 % overall yield, 96 % stereoselectivity; [Fig. 13]). The 26R-epimer (41) was then submitted to the previously described routine ([Fig. 12]) to yield (26R)-uncialamycin (11b). The comparison of the spectroscopic data of this compound with the isolated one confirmed the configuration of the natural uncialamycin as the 26R-epimer.

Fig. 13 Synthesis of (26R)-uncialamycin (11b).

If this synthetic strategy provided an answer to the question of the relative configuration at C26, it did not solve the issue of the absolute configuration of the natural product. To obtain more stereochemistry information, Nicolaou et al. developed a catalytic asymmetric synthesis of uncialamycin (11; [Fig. 14]) [55]. The prochiral quinoline carboxylic acid 42 was converted to its methyl ester 43 with a 65 % overall yield. Noyori reduction [75], [76] of the methyl ketone moiety within 43 resulted in the formation of γ-lactone 32, presumably via intermediate hydroxy ester 45, in a 95 % yield and 93 % ee. Conversion to compound 33, necessary for the pursuit of the synthesis [73], under acidic conditions led to difficulties in maintaining the configurational integrity of the generated asymmetric center. To reach compound 33, another route has been envisaged starting from compound 42. Methyl ether carboxylic acid 42 was converted to DMB ether 46 (55 % overall yield). Noyori reduction of 46 under the same conditions as those described previously for 43 furnished lactone 33 via intermediate 47, in a 95 % yield and 98 % ee. The intermediate 33 was then used to obtain (+)-uncialamycin (11, natural) and (+)-26-epi-uncialamycin as described for the synthesis of the racemic forms of these compounds [73].

Fig. 14 Catalytic asymmetric synthesis of chiral quinoline (11). DMAP: 4-(N,N-dimethylamino)pyridine; DMB: 3,4-dimethoxybenzyl; DMF: N,N-dimethylformamide; Ts: toluenesulfonyl.

One year after the realization of the first asymmetric synthesis of uncialamycin by Nicolaouʼs group, a second research group focused their interest on the synthesis of this metabolite. While Nicolaou and team introduced the asymmetry in their molecule almost at the end of their total synthesis, van de Weghe and coworkers based their approach on the preparation of a chiral quinoline by introducing the chirality from the second step of their synthetic strategy. They developed an intramolecular imino Diels-Alder reaction, based on the Povarov reaction, coupled to an oxidative aromatization that allowed the formation of substituted quinolones [77]. They used these reaction conditions to prepare the chiral quinoline moiety, as the chiral center C26 was fixed by an enantioselective reduction of an α-alkyne ketone 48 affording the alcohol 49 with 80 % ee. The enantiomeric excess has been determined by ¹H NMR after derivatization into its corresponding O-methylmandelic ester 56. The alkynol 49 was converted in acryloyl ester 51 and the diol 52 was isolated as a diastereomeric mixture after an osmylation reaction with a 55 % yield. The diol 52 was converted into its glyoxylic acid derivative and then transformed into the imine 53 ([Fig. 15]). After imino-Diels-Alder cycloaddition, the quinoline 54 was isolated in a 65 % yield overall from the diol 52 with an 89 % ee as determined by HPLC.

Fig. 15 Preparation of the chiral quinoline by P. van de Weghe and colleagues.

Later, van de Wegheʼs group developed a second approach for the total synthesis of uncialamycin [78]. [Fig. 16] shows the retrosynthetic analysis of the construction of the quinoline core. Fragment 58 was obtained from a Michael-aldolization-crotonization reaction cascade starting from compounds 60 and 61. The acetylide addition to the activated quinoline moiety followed by a ring closure reaction led to the synthon 57.

Fig. 16 Retrosynthetic approach developed by P. van de Weghe and colleagues.

The synthetic procedure started by the formation of the aniline derivative 63 using the Sugasawa reaction conditions [79]. This ortho-acylation has been achieved by condensation of chloroacetonitrile with compound 62, followed by a substantial HCl expulsion ([Fig. 17]). The aniline derivative 63 was submitted to a Michael addition with methyl vinyl ketone 61, which led to the Michael adduct 64. The aldolization of compound 64 has been successfully realized in the presence of Cs₂CO₃ under an oxygen atmosphere. The hemiketal 65 led to the ketone 66 in a 63 % yield over two steps [80], which was reduced into the racemic alcohol 67. At last, a protection step of the secondary alcohol of 67 afforded compound 68 as the quinoline key fragment. The quinoline core was activated using methyl chloroformate [81] and trapped by the addition of the acetylide derived from enediyne 69 to furnish 70 in a good yield as a mixture of cis/trans isomers in an unchanged ratio of 6 : 4, which were separated by flash chromatography. Each isomer was then submitted to a deprotection step followed by an epoxidation. Compound 70-cis gave a unique isomer of the corresponding oxirane, while 70-trans led to a mixture. Further experiments were performed using the epoxide from 70-cis. After oxidation of the primary alcohol, the cyclization step was carried out and yielded a mixture of 72 and 73 in a ratio of 7 : 3. After isolation, NMR spectroscopic data of 72 showed a consistent similitude with the quinoline/enediyne moiety of the uncialamycin 11.

Fig. 17 Synthesis of the synthon 72.

Conclusion

Lichens represent interesting microenvironments where a number of different organisms closely interact and have a rich bacterial diversity. Therefore, the lichen holobiont also has a very large potential for natural product discovery. So far, the bacterial diversity has been studied by various culture-dependent and culture-independent molecular and microscopic approaches, which in almost all of the studies highlighted the prevalence of Alphaproteobacteria, Firmicutes, and Actinobacteria. Various abiotic and biotic factors affect this diversity, and as a consequence, structure and localization of the bacterial microbiome may be correlated with chemical patterns of metabolites released by all symbionts in the lichen thallus. Even if many roles of these bacteria still need to be experimentally confirmed, it seems now clearer that they contribute to various functions such as protection against biotic and abiotic stresses, nutrient cycling, reallocation of resources, detoxification, and other functions. The lichen-associated culturable bacteria have become a promising source of interesting bioactive metabolites, with most of them exhibiting potent activities. To date, uncialamycin constitutes one of the most exciting compounds as it presents a broad variety of biological activities associated with a unique and specific mechanism of action. Two main approaches in the design of this compound have been undertaken and highlight the structurally complex enediyne derivatives as a challenging target for chemists. One of the strategies allowed for the determination of the configuration of the natural compound. These major breakthroughs paved the way for the synthesis of analogues and new related enediynes, which could become new pharmaceutical leads. Finally, these data confirm the real biotechnological interest of lichen-associated bacteria as an untapped source of interesting metabolites with useful activity. The next challenges will be to explore the whole metabolome of these bacteria by activating silent biosynthetic pathways. One of the approaches could be based on coculture studies.

Acknowledgements

This work was partly supported by EMR, a partnership between the UPMC, Laboratoires Pierre Fabre and the CNRS, by the project ANR MALICA (10-INBS-02–01), UFR Sciences Pharmaceutiques et Biologiques of Rennes for a grant for N. Legrave, as well as INSA, Rennes for the PhD grant of D. Parrot, Rennes Metropole, and PhD Amadeus for the exchange with Austria. We thank Nyree West, Laurent Intertaglia for fruitful discussions.

Conflict of Interest

The authors declare that they have no conflict of interest.

^* These authors contributed equally to this paper.

• References

• 1 Ling LL, Schneider T, Peoples AJ, Spoering AL, Engels I, Conlon BP, Mueller A, Hughes DE, Epstein S, Jones M, Lazarides L, Steadman VA, Cohen DR, Felix CR, Fetterman KA, Millett WP, Nitti AG, Zullo AM, Chen C, Lewis K. A new antibiotic kills pathogens without detectable resistance. Nature 2015; 517: 455-459
• 2 Lauterwein M, Oethinger M, Belsner K, Peters T, Marre R. In vitro activities of the lichen secondary metabolites vulpinic acid, (+)-usnic acid, and (−)-usnic acid against aerobic and anaerobic microorganisms. Antimicrob Agents Chemother 1995; 39: 2541-2543
• 3 Ingólfsdóttir K. Usnic acid. Phytochemistry 2002; 61: 729-736
• 4 Molnár K, Farkas E. Current results on biological activities of lichen secondary metabolites: a review. Z Naturforsch C 2010; 65: 157-173
• 5 Shrestha G, St Clair LL. Lichens: a promising source of antibiotic and anticancer drugs. Phytochem Rev 2013; 12: 229-244
• 6 Shukla V, Joshi GP, Rawat MSM. Lichens as a potential natural source of bioactive compounds: a review. Phytochem Rev 2010; 9: 303-314
• 7 Boustie J, Grube M. Lichens – a promising source of bioactive secondary metabolites. Plant Genet Ressources 2005; 3: 273-287
• 8 Boustie J, Tomasi S, Grube M. Bioactive lichen metabolites: alpine habitats as an untapped source. Phytochem Rev 2011; 10: 287-307
• 9 Hawksworth DL. The lichenicolous fungi of Great Britain and Ireland: an overview and annotated checklist. Lichenologist 2003; 35: 191-232
• 10 Lawrey J, Diederich P. Lichenicolous fungi: interactions, evolution, and biodiversity. Bryologist 2003; 106: 80-120
• 11 González I, Ayuso-Sacido A, Anderson A, Genilloud O. Actinomycetes isolated from lichens: evaluation of their diversity and detection of biosynthetic gene sequences. FEMS Microbiol Ecol 2005; 54: 401-415
• 12 Cardinale M, Puglia AM, Grube M. Molecular analysis of lichen-associated bacterial communities. FEMS Microbiol Ecol 2006; 57: 484-495
• 13 Cardinale M, Vieira de Castro jr. J, Müller H, Berg G, Grube M. In situ analysis of the bacterial community associated with the reindeer lichen Cladonia arbuscula reveals predominance of Alphaproteobacteria. FEMS Microbiol Ecol 2008; 66: 63-71
• 14 Cardinale M, Grube M, Castro jr. JV, Müller H, Berg G. Bacterial taxa associated with the lung lichen Lobaria pulmonaria are differentially shaped by geography and habitat. FEMS Microbiol Lett 2012; 329: 111-115
• 15 Liba CM, Ferrara FIS, Manfio GP, Fantinatti-Garboggini F, Albuquerque RC, Pavan C, Ramos PL, Moreira-Filho C, Barbosa HR. Nitrogen-fixing chemo-organotrophic bacteria isolated from cyanobacteria-deprived lichens and their ability to solubilize phosphate and to release amino acids and phytohormones. J Appl Microbiol 2006; 101: 1076-1086
• 16 Grube M, Berg G. Microbial consortia of bacteria and fungi with focus on the lichen symbiosis. Fungal Biol Rev 2009; 23: 72-85
• 17 Grube M, Cardinale M, de Castro jr. JV, Müller H, Berg G. Species-specific structural and functional diversity of bacterial communities in lichen symbioses. ISME J 2009; 3: 1105-1115
• 18 Hodkinson BP, Lutzoni F. A microbiotic survey of lichen-associated bacteria reveals a new lineage from the Rhizobiales. Symbiosis 2009; 49: 163-180
• 19 Schneider T, Schmid E, de Castro jr. JV, Cardinale M, Eberl L, Grube M, Berg G, Riedel K. Structure and function of the symbiosis partners of the lung lichen (Lobaria pulmonaria L. Hoffm.) analyzed by metaproteomics. Proteomics 2011; 11: 2752-2756
• 20 Bates ST, Cropsey GWG, Caporaso JG, Knight R, Fierer N. Bacterial communities associated with the lichen symbiosis. Appl Environ Microbiol 2011; 77: 1309-1314
• 21 Muggia L, Vancurova L, Škaloud P, Peksa O, Wedin M, Grube M. The symbiotic playground of lichen thalli – a highly flexible photobiont association in rock-inhabiting lichens. FEMS Microbiol Ecol 2013; 85: 313-323
• 22 Grube M, Cernava T, Soh J, Fuchs S, Aschenbrenner I, Lassek C, Wegner U, Becher D, Riedel K, Sensen CW, Berg G. Exploring functional contexts of symbiotic sustain within lichen-associated bacteria by comparative omics. ISME J 2015; 9: 1-13
• 23 Cardinale M, Steinová J, Rabensteiner J, Berg G, Grube M. Age, sun and substrate: triggers of bacterial communities in lichens. Environ Microbiol Rep 2012; 4: 23-28
• 24 Grube M, Berg G, Andrésson ÓS, Vilhelmsson O, Dyer PS, Miao VPW. Lichen genomics: prospects and progress. In: Martin F, Editor The ecological genomics of fungi. New York: John Wiley & Sons; 2014: 191-212
• 25 Printzen C, Fernández-Mendoza F, Muggia L, Berg G, Grube M. Alphaproteobacterial communities in geographically distant populations of the lichen Cetraria aculeata . FEMS Microbiol Ecol 2012; 82: 316-325
• 26 Bjelland T, Grube M, Hoem S, Jorgensen SL, Daae FL, Thorseth IH, Øvreås L. Microbial metacommunities in the lichen-rock habitat. Environ Microbiol Rep 2011; 3: 434-442
• 27 An SY, Xiao T, Yokota A. Leifsonia lichenia sp. nov., isolated from lichen in Japan. J Gen Appl Microbiol 2009; 55: 339-343
• 28 An SY, Xiao T, Yokota A. Schumannella luteola gen. nov., sp. nov., a novel genus of the family Microbacteriaceae. J Gen Appl Microbiol 2008; 258: 253-258
• 29 Cheenpracha S, Vidor NB, Yoshida WY, Davies J, Chang LC. Coumabiocins A–F, aminocoumarins from an organic extract of Streptomyces sp. L-4-4. J Nat Prod 2010; 73: 880-884
• 30 Davies J, Wang H, Taylor T, Warabi K, Huang XH, Andersen RJ. Uncialamycin, a new enediyne antibiotic. Org Lett 2005; 7: 5233-5236
• 31 Li B, Xie CH, Yokota A. Nocardioides exalbidus sp. nov., a novel actinomycete isolated from lichen in Izu-Oshima Island, Japan. Actinomycetologica 2007; 21: 22-26
• 32 Motohashi K, Takagi M, Yamamura H, Hayakawa M, Shin-ya K. A new angucycline and a new butenolide isolated from lichen-derived Streptomyces spp. J Antibiot (Tokyo) 2010; 63: 545-548
• 33 Selbmann L, Zucconi L, Ruisi S, Grube M, Cardinale M, Onofri S. Culturable bacteria associated with Antarctic lichens: affiliation and psychrotolerance. Polar Biol 2009; 33: 71-83
• 34 Williams DE, Davies J, Patrick BO, Bottriell H, Tarling T, Roberge M, Andersen RJ. Cladoniamides A–G, tryptophan-derived alkaloids produced in culture by Streptomyces uncialis . Org Lett 2008; 10: 3501-3504
• 35 Parrot D, Antony-Babu S, Intertaglia L, Grube M, Tomasi S, Suzuki MT. Littoral lichens as a novel source of potentially bioactive Actinobacteria. Sci Rep 2015; 5: 15839
• 36 Yamamura H, Ashizawa H, Nakagawa Y, Hamada M, Ishida Y, Otoguro M, Tamura T, Hayakawa M. Actinomycetospora iriomotensis sp. nov., a novel actinomycete isolated from a lichen sample. J Antibiot 2011; 64: 289-292
• 37 Suzuki MT, Parrot D, Berg G, Grube M, Tomasi S. Lichens as natural sources of biotechnologically relevant bacteria. Appl Microbiol Biotechnol 2016; 100: 583-595
• 38 Grube M, Köberl M, Lackner S, Berg C, Berg G. Host-parasite interaction and microbiome response: effects of fungal infections on the bacterial community of the Alpine lichen Solorina crocea . FEMS Microbiol Ecol 2012; 82: 472-481
• 39 Hodkinson BP, Gottel NR, Schadt CW, Lutzoni F. Photoautotrophic symbiont and geography are major factors affecting highly structured and diverse bacterial communities in the lichen microbiome. Environ Microbiol 2012; 14: 147-161
• 40 Mushegian AA, Peterson CN, Baker CMC, Pringle A. Bacterial diversity across individual lichens. Appl Environ Microbiol 2011; 77: 4249-4252
• 41 Sigurbjörnsdóttir MA, Heiðmarsson S, Jónsdóttir AR, Vilhelmsson O. Novel bacteria associated with Arctic seashore lichens have potential roles in nutrient scavenging. Can J Microbiol 2014; 92: 307-317
• 42 Navarro-Noya YE, Jiménez-Aguilar A, Valenzuela-Encinas C, Alcántara-Hernández J, Ruíz-Valdiviezo VM, Ponce-Mendoza A, Luna-Guido M, Marsch R, Dendooven L. Bacterial communities in soil under moss and lichen-moss crusts. Geomicrobiol J 2014; 31: 152-160
• 43 Davies J, Ryan KS. Introducing the parvome: bioactive compounds in the microbial world. ACS Chem Biol 2012; 7: 252-259
• 44 Williams DE, Bottriell H, Davies J, Tietjen I, Brockman MA, Andersen RJ. Unciaphenol, an Oxygenated Analogue of the Bergman Cyclization Product of Uncialamycin Exhibits Anti-HIV Activity. Org Lett 2015; 17: 5304-5307
• 45 Cardinale M, Grube M, Berg G. Frondihabitans cladoniiphilus sp. nov., an actinobacterium of the family Microbacteriaceae isolated from lichen, and emended description of the genus Frondihabitans. Int J Syst Evol Microbiol 2011; 61: 3033-3038
• 46 Singh S, Garrity G, Genillourd O, Lingham R, Martin I, Nallin-Omstead M, Silverman K, Zink D. Inhibitor compounds of farnesyl-protein transferase and chemotherapeutic compositions containing the same, produced by strain ATCC 55532. US Patent 5627057, 1997
• 47 Braña AF, Fiedler HP, Nava H, González V, Sarmiento-Vizcaíno A, Molina A, Acuña JL, García LA, Blanco G. Two Streptomyces species producing antibiotic, antitumor, and anti-inflammatory compounds are widespread among intertidal macroalgae and deep-sea coral reef invertebrates from the central Cantabrian Sea. Microb Ecol 2015; 69: 512-524
• 48 Takahashi Y, Omura S. Isolation of new actinomycete strains for the screening of new bioactive compounds. J Gen Appl Microbiol 2003; 49: 141-154
• 49 Murphy B, Jensen P, Fenical W. The chemistry of marine bacteria. In: Fattorusso E, Gerwick W, Taglialatela-Scafati O, editors Handbook of marine natural products. Dordrecht: Springer Netherlands; 2012: 153-190
• 50 Waters B, Saxena G, Wanggui Y, Kau D, Wrigley S, Stokesd R, Davies J. Identifying protein kinase inhibitors using an assay based on inhibition of aerial hyphae formation in Streptomyces . J Antibiot 2002; 55: 407-416
• 51 Yao G, Vidor NB, Foss AP, Chang LC. Lemnalosides A–D, decalin-type bicyclic diterpene glycosides from the marine soft coral Lemnalia sp. J Nat Prod 2007; 70: 901-905
• 52 Raad I, Hachem R, Abi-Said D, Rolston K, Whimbey E, Buzaid A, Legha S. A prospective crossover randomized trial of novobiocin and rifampin prophylaxis for the prevention of intravascular catheter infections in cancer patients treated with interleukin-2. Cancer 1998; 82: 403-411
• 53 Walsh TJ, Standiford HC, Reboli AC, John JF, Mulligan ME, Ribner BS, Montgomerie JZ, Goetz MB, Mayhall CG, Rimland D. Randomized double-blinded trial of rifampin with either novobiocin or trimethoprim-sulfamethoxazole against methicillin-resistant Staphylococcus aureus colonization: prevention of antimicrobial resistance and effect of host factors on outcome. Antimicrob Agents Chemother 1993; 37: 1334-1342
• 54 Konishi M, Ohkuma H, Tsuno T, Oki T. Crystal and molecular structure of dynemicin 1: a novel 1, 5-diyn-3-ene antitumor antibiotic. J Am Chem Soc 1990; 112: 3715-3716
• 55 Nicolaou KC, Chen JS, Zhang H, Montero A. Asymmetric synthesis and biological properties of uncialamycin and 26-epi-uncialamycin. Angew Chem Int Ed Engl 2008; 47: 185-189
• 56 Jean M, Tomasi S, van de Weghe P. When the nine-membered enediynes play hide and seek. Org Biomol Chem 2012; 10: 7453-7456
• 57 Damle NK. Tumour-targeted chemotherapy with immunoconjugates of calicheamicin. Expert Opin Biol Ther 2004; 4: 1445-1452
• 58 Berger M, Leopold L, Dowell J, Korth-Bradley J, Sherman M. Licensure of gemtuzumab ozogamicin for the treatment of selected patients 60 years of age or older with acute myeloid leukemia in first relapse. Invest New Drugs 2002; 20: 395-406
• 59 Hamann PR, Hinman LM, Beyer CF, Greenberger LM, Lin C, Lindh D, Menendez AT, Wallace R, Durr FE, Upeslacis J. An anti-MUC1 antibody-calicheamicin conjugate for treatment of solid tumors. Choice of linker and overcoming drug resistance. Bioconjug Chem 2005; 16: 346-353
• 60 Bergman RG. Reactive 1, 4-dehydroaromatics. Acc Chem Res 1973; 6: 25-31
• 61 Myers AG, Fraley ME, Tom NJ, Cohen SB, Madar DJ. Synthesis of (+)-dynemicin A and analogs of wide structural variability: establishment of the absolute configuration of natural dynemicin A. Chem Biol 1995; 2: 33-43
• 62 Smith AL, Nicolaou KC. The enediyne antibiotics. J Med Chem 1996; 39: 2103-2117
• 63 Sanchez C, Mendez C, Salas JA. Indolocarbazole natural products: occurrence, biosynthesis, and biological activity. Nat Prod Rep 2006; 23: 1007-1045
• 64 Howard-Jones AR, Walsh CT. Staurosporine and rebeccamycin aglycones are assembled by the oxidative action of StaP, StaC, and RebC on chromopyrrolic acid. J Am Chem Soc 2006; 128: 12289-12298
• 65 Hyun CG, Bililign T, Liao J, Thorson JS. The biosynthesis of indolocarbazoles in a heterologous E. coli host. Chembiochem 2003; 4: 114-117
• 66 Howard-Jones AR, Walsh CT. Enzymatic generation of the chromopyrrolic acid scaffold of rebeccamycin by the tandem action of RebO and RebD. Biochemistry 2005; 44: 15652-15663
• 67 Sánchez C, Méndez C, Salas J. Engineering biosynthetic pathways to generate antitumor indolocarbazole derivatives. J Ind Microbiol Biotechnol 2006; 33: 560-568
• 68 Sánchez C, Zhu L, Braña AF, Salas AP, Rohr J, Méndez C, Salas JA. Combinatorial biosynthesis of antitumor indolocarbazole compounds. Proc Natl Acad Sci U S A 2005; 102: 461-466
• 69 Zhang C, Albermann C, Fu X, Peters NR, Chisholm JD, Zhang G, Gilbert EJ, Wang PG, Van Vranken DL, Thorson JS. RebG- and RebM-catalyzed indolocarbazole diversification. Chembiochem 2006; 7: 795-804
• 70 Singh SB, Liesch JM, Lingham RB, Goetz MA, Gibbst JB. Actinoplanic acid A: a macrocyclic polycarboxylic acid which is a potent inhibitor of Ras farnesyl-protein transferase. J Am Chem Soc 1994; 116: 11606-11607
• 71 Silverman K, Cascales C, Genilloud O, Sigmund J, Gartner S, Koch G, Gagliardi M, Heimbuch BK, Nallin-Omstead M, Sanchez M, Diez M, Martin I, Garrity G, Hirsch C, Gibbs J, Singh S, Lingham R. Actinoplanic acids A and B as novel inhibitors of farnesyl-protein transferase. Appl Microbiol Biotechnol 1995; 43: 610-616
• 72 Fernandez-Medarde A, Santos E. Ras in cancer and developmental diseases. Genes Cancer 2011; 2: 344-358
• 73 Nicolaou KC, Zhang H, Chen JS, Crawford JJ, Pasunoori L. Total synthesis and stereochemistry of uncialamycin. Angew Chem Int Ed Engl 2007; 46: 4704-4707
• 74 Bretschneider H, Hohenlohe-Oehringen K, Rhomberg A. 3-acetyl-cinchoninic acid compounds. US Patent 3311632, 1967
• 75 Fujii A, Hashiguchi S, Uematsu N, Ikariya T, Noyori R. Ruthenium(II)-catalyzed asymmetric transfer hydrogenation of ketones using a formic acid−triethylamine mixture. J Am Chem Soc 1996; 118: 2521-2522
• 76 Noyori R, Hashiguchi S. Asymmetric transfer hydrogenation catalyzed by chiral ruthenium complexes. Acc Chem Res 1997; 30: 97-102
• 77 Desrat S, van de Weghe P. Intramolecular imino Diels-Alder reaction: progress toward the synthesis of uncialamycin. J Org Chem 2009; 74: 6728-6734
• 78 Desrat S, Jean M, van de Weghe P. Setbacks and hopes: en route to the synthesis of uncialamycin. Tetrahedron 2011; 67: 7510-7516
• 79 Prasad K, Lee GT, Chaudhary A, Girgis MJ, Streemke JW, Repič O. Design of new reaction conditions for the Sugasawa reaction based on mechanistic insights. Org Process Res Dev 2003; 7: 723-732
• 80 Bartoszewicz A, Kalek M, Stawinski J. Iodine-promoted silylation of alcohols with silyl chlorides. Synthetic and mechanistic studies. Tetrahedron 2008; 64: 8843-8850
• 81 Myers AG, Tom NJ, Fraley ME, Cohen SB, Madar DJ. A convergent synthetic route to (+)-dynemicin A and analogs of wide structural variability. J Am Chem Soc 1997; 7863: 6072-6094