History, biology and chemistry of Mycobacterium ulcerans infections (Buruli ulcer disease).

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History, biology and chemistry of Mycobacterium ulcerans infections (Buruli ulcer disease)† ´dric Tresse,a Virginie Casarottoa and Nicolas Blanchard*b Anne-Caroline Chany,a Ce

Covering: up to the end of July 2013 Mycobacterium ulcerans infections (Buruli ulcer disease) have a long history that can be traced back 150 Received 19th July 2013

years. The successive discoveries of the mycobacteria in 1948 and of mycolactone A/B in 1999, the toxin responsible for this dramatic necrotic skin disease, resulted in a paradigm shift concerning the disease

DOI: 10.1039/c3np70068b www.rsc.org/npr

1 2 3 4 5 6 6.1 6.2 6.3 6.4 6.4.1 6.4.2 6.4.3 6.4.4 6.4.5 6.4.6 7 7.1 7.2 7.3 7.4

itself and in a broader sense, delineated an entirely new role for bioactive polyketides as virulence factors. The fascinating history, biology and chemistry of M. ulcerans infections are discussed in this review.

A brief history of Mycobacterium ulcerans infections Clinical presentation and diagnosis of M. ulcerans infections Treatment of M. ulcerans infections The elusive reservoir of M. Ulcerans Mycolactone A/B, an unusual toxin Genome of M. ulcerans and biosyntheses of mycolactones The genome of M. ulcerans Reclassication of mycolactone-producing mycobacteria Biosynthesis of mycolactone A/B Domains and module alterations at the source of eight additional mycolactones Mycolactone C Mycolactone D Mycolactone E and its minor metabolite Mycolactones F and dia-F Mycolactones S1 and S2 Mycolactone G The biology of human M. ulcerans infections Cytotoxic effects of mycolactone A/B The localization of mycolactone A/B in cells Mycolactone A/B-mediated immunosuppression Mycolactone A/B-mimicking of endogenous regulators of WASP and N-WASP

8 8.1 8.2 8.2.1 8.2.2 8.2.3 8.2.4 8.3 8.4 8.5 9 9.1 9.2 9.3 9.4 9.4.1 9.4.2 9.4.3 10 11 12

Total syntheses of mycolactones A/B Overview of the synthetic strategies of Kishi, Negishi and Altmann Structural determination and total syntheses of mycolactone A/B by Kishi (2001–2010) Relative and absolute conguration (2001) First generation total synthesis (2002) Second generation total synthesis (2007) Third generation total synthesis (2010) Total synthesis of mycolactone A/B by Negishi (2011) Total synthesis of mycolactone A/B by Altmann (2011) Photochemical behavior of mycolactone A/B (2012) Synthesis of mycolactone analogues and structure– activity relationship studies Synthesis of mycolactone A/B analogues Synthesis of C8-desmethyl mycolactone A/B analogues Synthesis of uorescent mycolactone analogues Structure–activity relationship studies Naturally occurring mycolactones Chemical modications of naturally occurring mycolactone A/B SAR studies based on de novo chemical syntheses Conclusions Acknowledgements References

a

Université de Haute Alsace, Laboratoire de Chimie Organique et Bioorganique, EA4566, Ecole Nationale Supérieure de Chimie de Mulhouse, 3 rue Alfred Werner, 68093 Mulhouse Cedex, France

b Université de Strasbourg, Laboratoire de Chimie Moléculaire, CNRS UMR 7509, Ecole Européenne de Chimie, Polymères et Matériaux, 25 rue Becquerel, 67087 Strasbourg, France. E-mail: [email protected]; Tel: +33 3 89 33 68 24

† Electronic Supplementary Information (ESI) available: Total syntheses of mycolactones C, E, F, dia-F, S1 and S2. Partial syntheses of mycolactone A/B. See DOI: 10.1039/b000000x/

This journal is ª The Royal Society of Chemistry 2013

1 A brief history of Mycobacterium ulcerans infections In a seminal 1948 article in the Journal of Pathology and Bacteriology entitled “A new mycobacterial infection in man”, Peter MacCallum and three colleagues from the Pathology Departments of the Melbourne University and of the Alfred Hospital of

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Melbourne, Jean C. Tolhurst, Glen Buckle and Hubert A. Sissons described a series of six ulcerations with atypical histological and bacteriological characteristics.1 This rst clinical description of a devastating tropical disease now called Buruli ulcer, caused by the microorganism Mycobacterium ulcerans (M. ulcerans), was clearly a landmark discovery for numerous scientic elds, especially biomedical sciences. Another breakthrough was reported y years later in 1999 with the discovery of the M. ulcerans virulence factor, mycolactone A/B, an unusual macrolidic polyketide that has changed our perception of bioactive natural products. The impressive ulcers reported by MacCallum and coworkers were rst detected in 1940 on the leg of a two and half year-old boy who was admitted to a private hospital clinic of the farming district of Bairnsdale, a small city 280 kilometers east of Melbourne on the recommendation of Dr D. G. Alsop, a general practitioner (Fig. 1).2 The case history mentioned a dramatic evolution of the original infection, resulting in the eventual

death of the boy although no direct link to the ulceration was established: “in a period of six months, this ulcer denuded the limb laterally of skin and subcutaneous tissue down to the muscle over more than half the leg surface. It had a sloughing base and a hard, sharply cut, irregular edge and became foul smelling”.1 A biopsy was sent by Dr Alsop to the Department of Pathology of the University of Melbourne, which conrmed the discovery of highly unusual characteristics of the numerous acid-fast bacilli proliferating in this skin ulceration. These ulcers were primarily attributed to tuberculosis but the abundance, the grouping of the bacilli and the lack of a classical histological pattern of the tubercle attracted the attention of these Australian investigators (Fig. 2). Another striking feature of this organism is the difficulty to cultivate it on any type of media used for the tubercle bacillus and as stated in the original publication, “the suspicion that the organism was unusual and causally related to the ulcers was

Anne-Caroline Chany was born in 1983, studied chemistry at the Ecole Nationale Supérieure de Chimie de Mulhouse and obtained a Chemical Engineering degree and a Masters in 2008 aer having worked one year at Novartis in Vienna. She received her PhD in 2011 from the University of Haute-Alsace under the supervision of Nicolas Blanchard. Aer one additional year as a post doctoral researcher, working on the synthesis of bioactive natural products, she obtained a Marie Curie fellowship to work with Jonathan Burton at the University of Oxford on the development of new methodologies for the synthesis of complex natural products.

Virginie Casarotto received her PhD in December 2008 from the University of Toledo (USA), which she had joined in August 2003 as part of an exchange program with the Ecole Nationale Superieure de Chimie de Lille (France) from where she earned a Diplˆ ome d'ingénieur in September 2004. During her work on her PhD thesis, Virginie completed several studies on well-dened novel non-enzymatic catalysts and also worked on the total synthesis of ponicidin. In January 2009 she joined the group of Nicolas Blanchard to work on the synthesis of mycolactone analogs. Virginie is currently working as a clinical data manager at Quintiles Strasbourg.

Cédric Tresse was born in 1987 and studied chemistry at the University of Franche-Comté, Besançon, where he received his BSc degree in 2009. He then studied at the University of Orléans where he obtained his MSc degree. Aer a seven-month internship at Hoffmann-La Roche in Basel, Cédric moved to the University of Haute-Alsace in October 2011, where he is currently pursuing his PhD on the synthesis of mycolactone analogues under the supervision of Nicolas Blanchard.

Nicolas Blanchard was born in 1974, studied chemistry at the Ecole Normale Supérieure in Paris and received his PhD from the Université of Paris VI in 2000. Aer postdoctoral studies at Michigan University, he joined the CNRS as Chargé de Recherche in 2002 and became Directeur de Recherche at the University of Strasbourg in 2013. He has co-authored 60 publications, 4 book chapters and received the CNRS bronze medal (2012). Current research interests of his group encompass the development of efficient synthetic processes including metal and radical-mediated transformations and their applications in the synthesis of bioactive natural products.

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Fig. 1 1940 Day book entries of the Department of Pathology, University of Melbourne (Australia). Receipt of the first (top) and second (bottom) biopsies from Dr Alsop. Courtesy of Dr J. Hayman.

strengthened”. Actually, the cultivation of this new type of mycobacterium had puzzled the community for years as long series of failures were reported for different types of media, atmospheres and temperatures. It was serendipitously found that the only successful cultures had been realized in a defective incubator delivering a controlled 33 C. This breakthrough allowed Buckle and Tolhurst to establish the optimum conditions for the growth of the cultures between 30 and 33 C, although this growth is quite slow with a doubling time between thirty-six and eighty hours. It is worthy to note that this optimum temperature is consistent with the localization of the microorganisms within the subcutaneous fat and lower dermis of infected patients, as suggested by Buckle and Tolhurst in the original 1948 report1 and evidenced later on by Fenner in 1956.3 The microorganism was not named in the 1948 article but appeared as M. ulcerans two years later, in the footnote of a 1950 publication by Fenner.4 Australia was not the only country in which this necrotic disease was observed. In the late 1950s and 1960s, hundreds of cases of M. ulcerans infections were reported in Africa, mainly in the Belgian Congo by Janssens and coworkers5 and in Uganda by Clancey, Dodge and Lunn et al.6,7 Many of these observations were actually made in the early 1940s but the evidence for a new type of mycobacterial infection were so tenuous from a microbiological point of view that the scientic publications were postponed until the 1950s.8 The disease is now considered to be present, or at least suspected, in more than 31 countries on all continents. It is worth noting that the 1948 publication by MacCallum and coworkers, dening the infection in clinical terms, was preceded by a handful of scattered reports of large


Fig. 2 An artist's view of the cellular membrane covering the spleen of an infected rat, from the original 1948 publication by MacCallum and colleagues.1 Copyright ª 1948 The Pathological Society of Great Britain and Ireland.

ulcerations in Africa and Australia9 that were probable M. ulcerans infections and that could be traced back to the second half of the nineteenth century. The earliest report was made during the quest for the source of the white Nile in the 1860's, this discovery being coined “the greatest geographical secret, aer the discovery of America, which remained for the Caucasian's consideration” by the Victorian Africanist Sir Harry H. Johnston.10 In October 1860, an expedition initiated by the Royal Geographical Society of London and led by Captains John Hanning Specke and James Augustus Grant le Zanzibar “with the view to discover, if possible, the sources of the Nile”.11 Indeed, this expedition identied and named in July 1862 the Victoria lake and more precisely the Ripon falls at the northern end of the Lake, as the source of the white Nile. A detailed account of this fantastic odyssey was published by Grant in 1864 under the title “A walk across Africa or domestic scenes from my Nile journal”.12 Unfortunately for Grant, he was not on the shores of the Victoria lake in July 1862, as he got infected in December 1861 with a mysterious disease that led him to stay ve months bedridden.

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NPR The precise description of his illness is considered today as the rst report of a Buruli ulcer: “Having had fevers twice-a-month, in December [1861] my usual complaint assumed a new form. The right leg, from above the knee, became deformed with inammation, and remained for a month in this unaccountable state, giving intense pain, which was relieved temporarily by a deep incision and copious discharge. For three months fresh abscesses formed, and other incisions were made; my strength was prostrated; the knee stiff and alarmingly bent, and walking was impracticable. [.] By the h month, the complaint had exhausted itself; at last I was able to be out of the hut inhaling the sweet air, and once more permitted to behold the works of God's creation in the beautiful lake and hills below me. Never did I experience a happier moment!”.12b This succession of symptoms and slow healing aer a few months, leaving residual contractures, is consistent with an oedematous form that occurs in the Congo and is therefore the rst (probable) report of a M. ulcerans infection. In 1897, Sir Albert Cook described unusual ulcers in his case report from Kampala Hospital (Uganda) with an admission diagnosis of “tubercular ulceration of arms and legs”.13 This hospital also reported in 1910 the admission of a patient with an ulcerated septic leg (Fig. 3). The case history reports that the “whole leg and foot of the right side are swollen. Just below the knee on outer and post. aspect is a large raw deep and granulating ulcer”, another example of the oedematous form of the disease.14

Fig. 3 April 18th 1910, day book entry of the Mengo Hospital. Courtesy of Dr J. Hayman.

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Review The term “Buruli ulcer” was rst proposed by the investigators Clancey, Dodge and Lunn operating in Uganda, as most of their patients came from a semi-arid area called “Buruli” in the Mengo district of Uganda (the Nakasongola district since 1997).15 Cases of similar mycobacterial ulcerations were reported in the nearby Madi district, in which the local population used the term “juwe okoro” or “bile okoro” that could be translated by “the sore that heals in vain”.16 However, from an historical perspective, it is worth noting that this skin ulceration disease should be named “Bairnsdale ulcer” as pointed out by Radford as early as 1973,17 even though more than a dozen names have also been proposed throughout the literature.17a,18 Nevertheless, the terms “Buruli ulcer” or “Mycobacterium ulcerans infection” are now widely accepted for this necrotic disease and have been used interchangeably.19 “Buruli ulcer” also appears in the name of a World Health Organization's program launched in 1998, the Global Buruli Ulcer Initiative (GBUI) that promotes information and research efforts on this devastating but still neglected disease.20

2 Clinical presentation and diagnosis of M. ulcerans infections Although the prevalence of M. ulcerans infections is not clearly established, it is estimated that tens of thousands of cases have been reported in sub-Saharan Africa with highly variable prevalence rates that could reach 16% in some rural areas of Cˆ ote d'Ivoire, according to a 1995 study.21 In certain areas of Ghana, the Buruli ulcer disease is even the second most prevalent mycobacterial disease aer tuberculosis, with a prevalence of 66 per 100 000.22 The Buruli ulcer disease adopts different clinical forms that can be dramatic if le untreated, thus highlighting the need for an unambiguous method of diagnosis to avoid an excessive delay before a relevant treatment. It should be noted that common beliefs attributed this devastating skin disease to curses and witchcra,23 and that the church or the herbalists24 are usually the rst to be consulted in remote villages, therefore further delaying efficient and appropriate medical care. However, this over-simplied view has been recently challenged by a study in Cameroon, demonstrating that other structural elements (such as the effectiveness and cost of the treatment and the quality of the medical doctor to patient relationship) should also be taken into account.25 Early diagnosis of cutaneous non-tuberculous mycobacterial infections in general, and of Buruli ulcers more specically, is crucial for correct therapeutic management but is not trivial since thirty other mycobacterial species can cause cutaneous infections.26 Buruli ulcers can be diagnosed on clinical grounds27 but should be conrmed by laboratory procedures either via conventional or molecular assays of the cutaneous non-tuberculous mycobacterial infection. The rst clinical presentation of a Buruli ulcer is nonulcerative. An evolution is the ulcerative form, which is predominant in Africa (74%), Australia (87%) and Japan (94%) (Fig. 4).28,29 In the former presentation, papules (raised skin lesion with a diameter inferior to 1 cm), nodules (extension into


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Fig. 4 Buruli ulceration on the forearm of a 26-year-old male dairy-farm worker illustrating the original 1948 publication by MacCallum and colleagues.1 Copyright ª 1948 The Pathological Society of Great Britain and Ireland.

the subcutaneous tissue, diameter inferior to 2 cm), plaques (lesion with irregular edges and diameter superior to 2 cm, absent in Australia) and oedematous forms (diffuse swelling that can extend to all of a limb) can be observed. All these lesions are rm, painless and associated with color changes. Fever can also be observed. The more severe presentation of a Buruli ulcer, the ulcerative form, leads to large ulcerations with undermined edges and a white material can be observed on the oor of the ulceration. This still painless lesion can reach the bone and cause painful osteomyelitis or reactive osteitis.30 The World Health Organization, taking into account the size of the lesions, has introduced an additional classication into the three categories:29 category I concerns single lesions of less than 5 cm in diameter and that heal completely with an antibiotherapy (see Section 3), category II is for a single lesion measuring 5 to 15 cm in diameter that could potentially (but not always) be treated by antibiotics and the last category, III, is for extended single or multiple lesions, lesions at critical sites and cases of osteomyelitis. The therapeutic management of the latter category oen calls for surgery in addition to the classical antibiotherapy, as will be discussed in Section 3.29 It must be emphasized that the infection is localized mainly on the limbs (in 85% of cases) and can further reach muscles, blood vessels, nerves,31 bones and joints, leading to dramatic functional limitations.32 The mortality rate is low for M. ulcerans infections, however, the morbidity and associated socio-economic impact of the disease is a real burden for the population.33 Unambiguous identication of early M. ulcerans infections is quite challenging at the points of care owing to the large number of other non-tuberculous mycobacterial infections and the general level of technical equipment in endemic regions. The World Health Organization has therefore recommended that the clinically diagnosed or suspected cases should be conrmed by reference laboratories, either nationally or internationally. The classical sources of samples are pus or scrapings, biopsy samples, cultured samples and paraffin-embedded materials. The development of a minimally invasive technique for the sampling is highly desirable bearing in mind that 50% of


NPR the patients are children under 15 in Africa (10% in Australia and 19% in Japan).29 In this regard, preliminary studies based on ne-needle aspirates proved promising in particular for patients with non-ulcerative lesions.34 M. ulcerans stains red in the classical Ziehl–Neelsen test, indicative of an acid-fast bacilli. Conrmation of a Buruli ulcer is thus usually done by smear microscopy aer staining, which is not demanding but has only a low sensitivity. Recent studies have thus focused on the optimization of the release and concentration of the bacteria from swab specimens, leading to a sensitivity of 58.4% and specicity of 95.7%. An additional IS2404 PCR step could also be used (vide infra).35 Due to the low sensitivity of this test other assays are required. As outlined in Section 1, the cultivation of colonies is extremely time consuming, taking from six to eight weeks, since M. ulcerans is a slow growing organism (group II of the traditional Runyon classication), precluding cultivation as a rapid diagnostic method. In addition, the sensitivity of this assay is low, between 35 and 60%. However, it should be noted that cultivation is required for the monitoring of the antimycobacterial treatment.36 More denitive methods of diagnostics rely on molecular identication of cutaneous non-tuberculous mycobacterial infections. In this regard, PCR methods have become the gold standard for the denitive identication of Buruli ulcer infections, with a sensitivity superior to 96%.36–38 In particular, a dry reagent-based PCR (DRB-PCR) proved very reliable for the diagnosis of Buruli ulcers in the early stages and is well adapted to tropical conditions since its rst evaluation in 2003 at the Kumasi Centre for Collaborative Research in Tropical Medicine (Kumasi, Ghana).39 This PCR technique specically targets the insertion sequence 2404, a 1274 base pair-long element of the M. ulcerans genome, found only in this mycobacterium. Typically, the PCR-amplied sequences are less than 200 base pairs. The sensitivity of the IS2404 PCR is very high since a 0.1 M. ulcerans genome equivalent could be detected.40 Recent evolution has included, for example, the development of a 16S rRNA reverse transcriptase/IS2404 real-time quantitative PCR that allows for the quantication of M. ulcerans.41 However, PCR techniques are expensive and rely on highly qualied technical staff, conditions that are present at well-equipped reference laboratories but not in rural endemic regions. Collected samples thus need to be shipped to reference laboratories, which are either national or international, with a strict respect of cold conditions. Logically, the implementation of national Buruli ulcer disease reference laboratories with high standards are encouraged and such an example was recently reported for Togo.42 A recent and very promising evolution of the DNA amplication strategies is loop isothermal amplication (LAMP), which is able to specically amplify DNA from M. ulcerans but not from other Mycobacterium species, with ten times higher sensitivity than the IS2404 based PCR method.43 In addition, this method is cost- and time-effective as puried DNA samples are not a requirement, making it theoretically amenable to the remote point of care facilities. Further evaluation of the Buruli ulcer-LAMP diagnostic is required on larger sets of freshly collected samples.

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NPR A last diagnostic method based on the detection of mycolactone A/B, the causative agent of Buruli ulcers that will be discussed extensively in the next sections, should be mentioned. Mycolactone A/B is the virulence factor produced by M. ulcerans and was shown to diffuse beyond the foci of primary infection, making it an interesting target to be detected in circulating blood cells.44 By classical thin layer chromatography, mycolactone A/B could be detected up to 20 to 30 ng. An insightful method for lowering this limit of detection was reported by Kishi and collaborator using a uorescence enhancer (Fig. 5).45 Upon complexation of the 1,3-diol units of mycolactone A/B with 2-naphthylboronic acid, two cyclic boronates are formed. Irradiation at 365 nm led to a uorescence emission from the pentaenoate around 520 nm, that is greatly enhanced by the C13,C15-cyclic boronate. With the limit of detection of mycolactone A/B being 2 ng with synthetic and pure material, this method is very sensitive and holds great promises for a practical method of detection of the Buruli ulcer toxin, as was demonstrated on lipidic extracts from human skin biopsies.46 However, it should be noted that samples collected from patients' serum and ulcer exudates could also be contaminated with various human lipids and that their auto-uorescence could hamper the clear identication of mycolactone A/B.47 In addition, the toxin was only slowly eliminated, even aer antibiotic therapy, suggesting that the

Fig. 5 Experimental protocol. Step a: an acetone solution of mycolactone A/B (10 ng) is applied to a dye-free silica gel C60 TLC plate and eluted with 90 : 9 : 1 CHCl3–MeOH–H2O as the mobile phase; mycolactone A/B is not detected with conventional methods (photograph I, top). Step b: the eluted TLC plate is briefly warmed on a hot plate to evaporate the solvents and, while warm, is quickly immersed into a 0.1 M acetone solution of 2-naphthylboronic acid. Step c: the TLC plate is heated to 100 C for 5–10 s. Step d: the TLC plate is irradiated with a UV lamp (8 W) with a 365 nm filter, after its reverse side has been cleaned with acetone, to detect mycolactone A/B as a green-yellow fluorescent spot (photograph II, bottom). Scheme and Caption reproduced from Ref. 45 with permission from The Royal Society of Chemistry.

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Review detection of mycolactone A/B will not be relevant for the therapeutic monitoring of this dramatic disease.47

3

Treatment of M. ulcerans infections

Besides the rare spontaneous cases of healing that were reported in the literature, surgery was historically the method of choice for the management of the Buruli ulcer disease with removal of all infected tissue, followed by skin graing.26 However, this treatment was really drastic, involving long hospitalization (an average of three months) for rural areas that were already lacking hospital bed capacity. In addition, the recurrence rates were between 16% and 28%. The revolution in the treatment of Buruli ulcers came in 2004 with the introduction of antibiotherapy, which reduced the recurrence rate to between 0 and 2%, and also diminished the use of surgery.26,29,48 It is beyond the scope of this review to cover in a comprehensive manner the numerous classes of drugs (such as aminoglycosides, macrolides, quinolones) or alternative treatments (such as natural products,49 hyperbaric oxygen,50 bacterial viruses51) that have been proposed52 for the control of this terrible skin disease and we will focus only on the currently optimized treatment, i.e. oral rifampin and intramuscular streptomycin, a combination necessary to avoid the selection of drug-resistant mutants of M. ulcerans, administered daily for eight consecutive weeks. This antibiotherapy is now widely used by the experts (although some paradoxical reactions were observed in rare cases)53 for all categories of ulcers, even for oedematous lesions. From a mechanistic point of view, it is suggested that this combination of drugs blocks the production of mycolactone A/B and kills M. ulcerans, although no detailed studies are currently available.54 For large ulcerations, however, antibiotics should be accompanied in a second stage by surgery (debridement and skin graing) to facilitate healing. In order to further tune the treatment, trials using two to four weeks of rifampin/streptomycin were followed by four to six weeks of fully oral antibiotherapy with rifampin and clarithromycin55 or rifapentine/clarithromycin.56 These preliminary studies are very encouraging and might contribute to an even more practical therapeutic management of Buruli ulcer disease in the near future. Recently, attention has also been drawn to secondary bacterial infections that could further delay a complete recovery of the infected patients.57 Besides antibiotherapy, the general wound care is also of the utmost importance to limit future functional and movement restrictions. This is likely to occur when the foci of infection are near wrists, ankles, elbow or when the bandaging prohibits easy movement.32 It should be mentioned that there is no vaccine currently effective against M. ulcerans infections, although a cross-reactive protective role of the BCG vaccine was observed transiently.58 The development of a specic and efficient vaccine is thus a crucial endeavor that started y years ago,59 and which is still a vibrant area of research.60 As a nal note, inhibiting the biosynthesis of mycolactone A/B has also been proposed as an alternative strategy for the therapeutic management of Buruli ulcer infections, with limited results up to now.61


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4

The elusive reservoir of M. Ulcerans

The natural reservoir of M. ulcerans is still an open question aer decades of investigation.62 Early studies in Africa demonstrated that the infection occurred near slow-moving water, or areas surrounded by water.7,8,15,16 The hypothesis of an aquaticrelated reservoir was therefore plausible and supported by the fact that M. ulcerans assembles itself into a protein-rich biolm, rst observed on aquatic plants from the Scrophulariaceae family collected in 2001 from the Lobo River in Cˆ ote d'Ivoire.63 This biolm contains an abundant extracellular matrix that is one of the reservoirs of mycolactone A/B and that also increases dramatically the colonization potency of the mycobacterium.64 Actually, predaceous aquatic insects, such as those belonging to the genus Naucoris are able to host the mycobacteria in the salivary glands without incidence on their own tissues and could transmit M. ulcerans to laboratory mice through bites.65 The central role of the extracellular matrix in this colonization phenomenon was demonstrated. Recently, increasing evidence that sh and amphibians could also act as a potential reservoir were reported, the latter constituting a missing link between aquatic and terrestrial environments.66 Mammals,67 such as the marsupials Pseudocheirus peregrinus and Trichosurus vulpecula, as well as mosquitoes68 were also proposed as hosts of M. ulcerans in Australia, although in the latter case the fact that mosquitoes were active vectors of the infection has been challenged.69 As an example, multiple modes of transmission were recently proposed in a study of patients from the Map´ e basin of Cameroon since the M. ulcerans lesions were clustered around the ankles and the elbows, a fact that is not consistent with the classical pattern of mosquito bites.70 In any case, the production of mycolactones by M. ulcerans could be an evasion strategy conferring a decisive advantage for survival in a new host and/or new environment.

5

Mycolactone A/B, an unusual toxin

One of the most intriguing facts concerning Buruli ulcer infections is that the proliferation of these extracellular acidfast bacilli, “grouped characteristically in sharply dened oval or rounded masses”,1 is maximal in the center of the necrotic lesion, which led Connor and Lunn8 to propose the existence of a diffusible toxin, possibly an enzyme as noted by Rees,8b produced by the mycobacteria, as early as 1965. This hypothesis, in hindsight, was truly visionary, as we will see in this section. The nature of the toxin produced by M. ulcerans remained elusive for several decades although hints were regularly reported. In 1974, Krieg, Hockmeyer and Connor reported that this toxin was present in three fractions of M. ulcerans cultures from Zaire: the culture ltrate, the particulate fraction and the cytoplasmic uid, while being excluded from the cell-wall fraction.71 A cytophatic activity effect (CPE), namely the rounding up of adherent cells followed by cell detachment aer 48– 72 h from the culture plate as a measure of cytotoxicity, was observed when L929 tissue cells were inoculated with these three fractions. Inammation and skin destruction in guinea


NPR pigs were also noted upon intradermal inoculation. Further investigations by the same group led to the hypothesis that this toxin was a “protein in association with a lipid and/or a polysaccharide or possibly a mixture of several toxin molecules”.72 However, this was in contradiction with the fact that heating the sterile ltrate containing the toxin at 100 C for one hour led to the retention of the cytotoxic activity.72 The phospholipoprotein–polysaccharide complex nature of the toxin was nally rejected in 1998 by George and collaborators, who showed that the sterile ltrate was protease resistant and that the acetone soluble fraction of the M. ulcerans ltrate was able to induce a signicant CPE on L929 cells as a consequence of dramatic cytoskeletal rearrangement.73 Eventually, Small and collaborators reported the structure of this toxin in 1999 and its role in the development of M. ulcerans infections was unambiguously demonstrated.74 The report by Small et al. was based on the cultivation of M. ulcerans for four to six weeks at 32 C. The cell aggregates were dried to give 1.4 g of cell weight per liter75 and then an icecold acetone soluble fraction of this crude extract was partially puried by thin-layer chromatography followed by reverse phase HPLC. The yellow glass thus obtained (4–6 mg per liter)75 was subjected to extensive high-resolution mass spectrometry, infra-red and multinuclear NMR spectroscopic investigations.76 Quite surprisingly, the toxin proved to be a complex polyketidic undecenolide substituted at C11 by a ten-carbon atom chain (northern fragment) and at C5 by a pentaenoic acid ester (southern fragment) (Fig. 6). This macrolide was named mycolactone by Small et al. to highlight both its mycobacterial origin and macrolidic nature. Mycolactone exists as a dynamic equilibrium of two geometrical isomers in the C40 –C70 dienic motif, the Z-isomer (mycolactone A) being predominant due to severe allylic strain77 in the corresponding E-isomer (mycolactone B) 0 0 0 0 (Z-D4 ,5 /E-D4 ,5 ¼ 60 : 40). Mycolactones A and B could be separated by reverse phase HPLC but spontaneously re-isomerize to the 60 : 40 thermodynamic equilibrium,76 even when protected from light. Protection against light is important for the toxin's integrity, since the light-induced degradation of mycolactones A/B has been reported by Marion and Marsollier et al. at a number of wavelengths (254, 312 and 365 nm, visible light such as sunlight, incandescent and uorescent bulbs).78 However, it must be kept in mind that the phototherapy of Buruli ulcers by

Fig. 6

0

0

0

0

Mycolactone A/B as Z-D4 ,5 /E-D4 ,5 ¼ 60 : 40 dynamic equilibrium.

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degradation and/or photoconversion of mycolactone is conceptually inadequate since the ulcer presents undermined edges that protect the toxins from light. The structure of the UVinduced photoproducts of mycolactones A/B has been elegantly established by Kishi et al. and will be presented in Section 8.5 in detail.79 The landmark discovery of mycolactones A/B has shown for the rst time that complex polyketidic macrolides could exist in pathogenic mycobacteria and that they could be virulence determinants for a variety of hosts, including humans. This paradigm shi was immediately appreciated by the community, as it constituted the rst evidence of a new role for bioactive polyketides.74,80 Quite logically, the study of the genome and of the biosynthesis of these unusual polyketides attracted a lot of attention, as discussed in the next section.

6 Genome of M. ulcerans and biosyntheses of mycolactones 6.1

The genome of M. ulcerans

As mentioned in Section 5, the discovery of the cytotoxic mycolactone A/B clearly delineated a new role for polyketides as potential human pathogens.74,80 Polyketides represent an important family of natural products oen involved in secondary metabolism, and present interesting biological activities that could even transform them into very useful therapeutic or agrochemical agents such as the well known erythromycin, FK-506 or the avermectins.81 The elucidation of the biosynthesis of this family of natural products has triggered numerous studies over the past few decades and it is now well established that enzymes called polyketide synthases (PKS) are involved, via the controlled assembly of simple subunits such as acetyl-CoA or malonyl-CoA, in the synthesis and functionalization of long polyketidic chains.82 In the eld of mycolactones, a decisive step-forward was reported in 2004 with the complete decoding of the genome of M. ulcerans by Cole and coworkers,83 thereby opening the way to a new understanding of the origin and structural diversity of this family of toxins. Comparative genomic analysis showed that M. ulcerans evolved, one million years ago, from M. marinum by horizontal gene transfer and reductive evolution. These two strains share a 98% identical DNA sequence84 but do not exhibit the same phenotype.85 M. marinum, which causes granulomatous intracellular lesions, grows fast and produces carotenoid pigments to protect itself from light, but does not produce mycolactone. On the other hand, M. ulcerans grows slowly (doubling time between thirty-six and eighty hours), at lower temperature (30–33 C), does not produce carotenoid pigments and causes extracellular lesions.84 The M. marinum genome is composed of 6.6 Mbp, 5426 protein-coding genes and 65 pseudogenes, whereas the genome of M. ulcerans is composed of 5.8 Mbp, 4160 protein-coding genes and 771 pseudogenes.84,86 Early in this evolution, a 174-kb virulence plasmid named pMUM001 was acquired by M. ulcerans (Fig. 7).83,87 Quite importantly, this virulence plasmid acquisition certainly allowed M. ulcerans to adapt to a new

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environment,88 a hypothesis that is further supported by the fact that the M. ulcerans and M. marinum genomes are very close. More than half of this pMUM001 plasmid encodes for PKS proteins of unprecedented size, responsible for the mycolactone biosynthesis as suggested by genomic subtraction and proven by DNA sequencing and transposon mutagenesis experiments.89 A massive expansion of insertion sequences (IS) was also reported.84,90–92 Ten IS were discovered in the genome of M. ulcerans, including 213 copies of IS2404 (1274 bp),88 specic to M. ulcerans and 91 copies of IS2606 (1368 bp), (found also in M. lentiavum, but genetically distinct from M. ulcerans), which disrupt more than 110 genes.87 Indeed IS are well known to promote genome rearrangement and therefore the formation of pseudogenes (inactivated genes).93 The genome of M. ulcerans therefore contains a large number of pseudogenes and also two prophages (phiMU01 and phiMU02). The crtI gene, responsible for biosynthesis of carotenoid pigments, is shorter or inactive, which could be triggered by the fact that M. ulcerans probably lives in a dark environment.88 Genome rearrangement and gene deletions were also observed between these two strains, leading to the loss of approximately 1.1 Mbp.88 As an example, the function of several highly immunological mycobacterial proteins encoded by the ESX (esxA and esxB) and hspX genes, were disrupted or completely lost, which could be an advantage for mycolactone producers to adapt to a new environment and/or a new host possessing immunological defense mechanisms.94 6.2

Reclassication of mycolactone-producing mycobacteria

Besides its beautiful chemical structure, the most outstanding feature of the mycolactone family is a structural heterogeneity conned to the poly-unsaturated south chain while the twelvemember macrolactonic ring and the north chain are conserved (see Section 6.4),95 a specicity that could be explained by variations of the appropriate polyketide synthase, as discussed in the previous sections. As will be discussed in Section 6.4, the

Fig. 7 The 174-kb virulence plasmid pMUM001 of M. ulcerans.83 Copyright ª 2004 by the National Academy of Sciences.


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fact that several types of mycobacteria were producing different mycolactones that are capable of infecting a broad range of hosts is a very intriguing feature that has hampered the early recognition that all these mycobacteria should be considered as a single species. Actually, several names for the strains of mycolactone-producing mycobacteria (MPM) have been proposed, M. marinum, M. pseudoshottsii, M. shinshuense and M. liandii, a quite confusing nomenclature. Stinear and colleague recently proposed that all MPM should be renamed M. ulcerans, a taxonomically correct designation that is further supported by an extensive comparative genomic analysis as discussed in Section 6.1.96,97 These MPM evolved from a common M. marinum progenitor but not at the same rate, which depended on their adaptation to a new environment. Three distinct lineages have been characterized in studies of the Single Nucleotide Polymorphism (SNP) of several strains.96b The lineage 1 includes haplotypes from human strains of South America, Asia and Mexico, and also strains from sh and frog pathogens, whereas the lineage 2 contains the strain from Japan. These two lineages belong to the “ancestral lineage”.98 Lineage 3, also named “classical lineage” includes haplotypes from human and animal pathogens from Africa, Australia and South East Asia.98 The ancestral lineages (1 and 2) diverged from the classical lineage about 400 000 years ago and are genetically closer to M. marinum.84,88,99 These lineages seem to be less virulent but not less pathogenic than the classical lineage.98,107 A plasmid was also acquired by the MPM as well as several IS.100 As an example, IS2404 was acquired before the dispersion of these strains all over the world but IS2606 does not exist in all mycobacterium and was independently acquired by the lineages.92 The African and Australian strains contain the most important number of IS2606, indicating a possible impact of the highest number of this IS on the virulence or on the adaptation to a new environment.96b,101 On the other hand, lineage 3 has undergone a faster evolution to adapt to a new environment and most of the Buruli ulcer infections are due to strains from lineage 3. Based on these genomic elements and as mentioned earlier, a recent reclassication of the MPM as a single species, M. ulcerans, has been proposed to avoid confusing nomenclature.96

6.3

Fig. 8 A schematic representation of mycolactone A/B biosynthesis based on the complete sequence of pMUM001. KS: ketosynthase; DH: dehydratase; ACP: acyl carrier protein; AT1: acyltransferase 1 (malonate); AT2: acyltransferase 2 (malonate); AT3: acyltransferase 3 (methylmalonate); KRA: ketoreductase type A; KRB: ketoreductase type B; ER: enoylreductase.


Biosynthesis of mycolactone A/B

Proteomics studies demonstrated that the biosynthesis of the mycolactone, encoded in the giant plasmid pMUM001, seems to be located at or near the membrane, which could help mycolactone export.102 More specically, pMUM001 is present at the level of 1.9 copies per cell, contains 81 CDs (protein-coding sequences) and 26 insertions sequences including four copies of IS2404 and 8 copies of IS2606.87 Most of this giant plasmid consists of three genes, mlsA1, mlsA2 and mlsB encoding for three type I PKS of unprecedented size, MLSA1 (1.8 MDa), MLSA2 (0.26 MDa) and MLSB (1.2 MDa). The transcription of these PKS is done by a novel SigA-like promoter sequence.103 MLSA1 and MLSA2, composed of a loading module, nine extension modules and a terminal thioesterase (TE), produce

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the C1–C20 fragment of the mycolactones.83,89 MLSB consists of one loading module, seven extension modules and one thioesterase, accounting for the C10 –C160 side chain synthesis. Each module of the different domains of MLSA and MLSB contains several enzymes (ketosynthase KS, acyltransferase AT, keto-reductase KR, dehydratase DH and enoyl reductase ER) allowing the synthesis and the functionalization of the polyketidic chains.82 The malonyl- and/or methylmalonyl units are xed on the thiol residue of the Acyl Carrier Protein (ACP) and a decarboxylative condensation reaction is catalysed by the

Fig. 9

loading module. The units thus obtained can navigate downstream through the different modules thanks to the acyltransferases and can undergo functional adjustments like keto-reduction or dehydration reactions (Fig. 8). Three types of acyltransferases are present on this locus, two with malonate specicity (AT1 and AT2) and the third one with methylmalonate specicity (AT3). The assembly of the units is repeated until the required length is obtained. The formation of the alcohol functionality in C17 of mycolactone A/B, done in the module 2 of MLSA1, is worthy comment. From the chemical

The mycolactone family (1999–2012).

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Review logic point of view, it should be noted that the different domains of module 2 imply the reduction of an a,b-unsaturated carbonyl derivative that should lead to a C17-deoxy mycolactone A/B. This reductive domain is therefore certainly nonutilized as pointed out by Cole and coworkers,83 a phenomenon that has already been observed in the rapamycin-producing polyketide synthase.104 The synthesis of the C1–C20 fragment ends by the cyclization reaction to the desired undecenolide thanks to the MLSA2 thioesterase. The latter has been cloned and expressed and was shown to induce the formation of the undecenolide via a unique mechanism, quite different in its kinetic and inhibitory prole from the classical PKS thioesterases.61 On the other hand, the C10 –C160 chain is prepared thanks to MLSB. However, it should be noted that this southern side chain is not yet hydroxylated at C120 (vide infra). The C10 activated acyl-enzyme intermediate is then esteried with the C5-hydroxyl group of the C1–C20 macrolactonic fragment. It has been proposed that this step involved the MUP_045

NPR accessory gene of the PKS locus of pMUM001, coding for an enzyme that seems to be a type III “FAbH-like” ketosynthase, although the activity of mup045 has not yet been demonstrated. Finally, the hydroxy group at C120 is introduced in a post-PKS step thanks to the MUP_053 accessory gene encoding for a cytochrome P450 hydroxylase (cyp140A7).109 The absolute conguration of the different stereogenic centers is controlled during the reduction of the corresponding ketones. Ketoreductase modules exist as two types, KRA and KRB, where A and B correspond to the selectivity of the reduction, itself correlated with the orientation of key amino-acid residues in the enzyme active site.82g,105,106 The acyltransferases 1 or 2 of module 5 in MLSA1 and of module 1 and 2 in MLSB are linked with a KRA domain. HPLC analysis of mycolactone A/B isolated from several strains of M. ulcerans (1615, Agy 99, 1059, 1062) conrms that all mycolactones present the same absolute conguration and therefore that all these mycobacterium contain the same pMUM001 plasmid.

Fig. 10 Genetic organisation of the mycolactone biosynthetic cluster from pMUM001, pMUM002 and proposed organization for pMUM003. Mycolactone PKS module and domain structure is outlined, with the figure key showing the type of domains present in each of the modules. The mycolactone modules are color coded based on the type of module responsible for the addition of each two-carbon unit. Shaded modules indicate that the DNA sequence of these regions is unknown or not yet confirmed. *Organisation of mlsA1 and mlsA2 for all pMUM examined to date, based upon toxin structures. Figure and caption reproduced from Ref. 107.


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NPR These PKS of unprecedented size possess a high level of sequence identity through the different domains, thus conferring a high potential for recombination. However, besides mycolactone A/B, only eight other mycolactones have been isolated up to now from different strains and only due to modication on MLSB, as will be discussed in the next section. 6.4 Domains and module alterations at the source of eight additional mycolactones Isolation and structural determination of mycolactone A/B preceded the discovery of eight other members of this seducing toxin's family (C, D, E plus its minor metabolite, F, dia-F, S1 and S2) that were isolated as major or minor species from Malaysian, Western African, Australian, Chinese and Japanese human strains as well as from sh and frog pathogens (Fig. 9).75,107–109 All these mycolactones share a common C1–C20 fragment and differ only by the C10 –C160 fragment. In addition, these macrolides are present as a dynamic equilibrium of (at least) two geometrical isomers in the fatty acid side chain. This fact was recognized in the rst discovered mycolactone with two letters 0 0 0 0 (A/B) for the two isomers (Z-D4 ,5 /E-D4 ,5 ), but only a single letter (C, D, E, F, S) was used later on. 6.4.1 Mycolactone C. Mycolactone C was reported in 2003 as the major component of eight Australian strains and a minor component of Malaysian and Benin isolates.108 This strain is decient in the MUP_053 accessory gene encoding for the P450 cytochrome cyp140A7 (Fig. 10), responsible for the post-PKS hydroxylation of the C120 position of the fatty acid side chain (vide supra). This C120 -deoxy mycolactone A/B was named mycolactone C and its structure was deduced from high resolution mass spectrometry experiments and further conrmed by total synthesis,110 as presented in detail in the ESI.† The biological activity of mycolactone C was also investigated and it was shown that the cytopathicity on L929 broblasts was 15.5 times inferior to mycolactone A/B, with a lethal dose 50 (LC50, the concentration of mycolactone for which half of the cells were killed) of 186 nM.111

Fig. 11 Bacterial colonies of M. ulcerans ecovar Liflandii (clockwise from the top: M. ulcerans 1615 then M. ulcerans ecovar Liflandii).115 Reproduced with permission from the American Society for Microbiology.

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Review 6.4.2 Mycolactone D. Mycolactone D was rst mentioned in 2003 by Small et al. as the major toxin from two Asian strains that were collected in 1980 in Japan (strain 8756) and in 1997 in China (strain 98-912), although its structure was misleadingly attributed to a “congener with an additional oxygen atom”.108 Thanks to LC-sequential mass spectrometry (LC-MSn), Leadlay and coworkers disclosed the structure of mycolactone D as the C20 -methylated analogue of mycolactones A/B. This structural variation originates from a switch in the MLSB module 7 acyltransferase from an AT1 (malonate) to an AT3 (methylmalonate) (Fig. 10).121 The absolute conguration of this mycolactone is still unknown. 6.4.3 Mycolactone E and its minor metabolite. Mycolactone E was isolated from a different pathogenic agent, a M. ulcerans-like mycobacteria rst called (unofficially) M. liandii or more recently M. ulcerans ecovar Liandii112 that caused an epizootic mycobacteriosis of a colony of an African tropical frog named Xenopus (Silurana) tropicalis at the University of California, Berkeley in 2001–2002.113 This new member of the mycolactone family presents a diminished cytopathic activity compared to the A/B and thanks to MS-MS analysis114,115 and total synthesis,116 proved to be structurally different in the length, number of unsaturations and number of hydroxyl groups in the southern fragment. It is interesting to note that the tetraenoate motif confers an orange aspect to the mycobacterial colonies instead of the classical bright yellow observed for colonies producing mycolactones A/B (Fig. 11).114 Early studies have also established that mycolactone E is 100 times less cytopathic than mycolactone A/B on L929 broblasts.115 During mass spectroscopic investigations of the cell extract, Leadlay and colleagues showed that the mycolactone E characteristic ion (m/z 737) was accompanied by less than 10% of another molecular species at m/z 735 whose fragmentation, and notably the loss of a 2-butanone motif via a McLafferty rearrangement, suggested a C13-keto mycolactone E.114 The structure of this minor metabolite from M. ulcerans ecovar Liandii was conrmed by Kishi et al. through an elegant synthetic approach combined with comparison of HPLC proles (see ESI†).117 6.4.4 Mycolactones F and dia-F. Besides humans and frogs, mycolactones were discovered to infect sh with a worldwide distribution when produced from another type of mycobacteria, M. marinum. In 1990, Colorni and coworkers reported on a mycobacteriosis in Dicentrarchus labrax, a european sea bass cultured in Eilat, Red Sea, Israel. This infectious disease has now been found in more than twenty sh species of marine and freshwater origins and even in a hawksbill sea turtle.118 M. marinum is also able to cause human cutaneous lesions as granulomatous diseases were observed in aquarists and professional sh breeders.119 Structural studies were conducted by Small and colleagues on several strains of mycobacteria, especially on M. marinum DL240490 isolated from a sea bass. Ion trap MS/MS analysis of the acetone-soluble lipidic extracts followed by reverse phase HPLC and multinuclear NMR studies led to the identication of a novel mycolactone, coined mycolactone F by Small et al., having a conserved twelve-membered


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Review lactone but a shorter polyenic south chain with only two hydroxyl groups.120,121 As pointed out by the authors, “all strains produce the same molecule. However, differences in stereochemistry, though unlikely, cannot be ruled out”.120 Indeed, nature is quite malicious and insightful studies by Kishi and collaborator demonstrated that the absolute stereochemistry of the fatty acid side chain of mycolactone F isolated from D. labrax, a saltwater sh, was opposite to the one of mycolactones A–E (see ESI†).122 The biological activity of mycolactone F was also investigated and it was shown that the cytopathicity on L929 broblasts was 2.4 times inferior to the one of mycolactone A/B, with an LC50 of 29 nM.111 On the other hand, mycolactone dia-F was reported to be 1000 times less active than mycolactone F. 122 Quite surprisingly, stereochemical heterogeneity was found between the mycolactones produced by the mycobacteria infecting saltwater and freshwater sh. Initial studies by the Small group were based on nine strains, from which only two were from cultured freshwater sh (strain CC240299 from Koi and BB170200 from a silver perch).120 The latter strains were studied in detail by Kishi and coworkers using a combination of total synthesis and chiral HPLC methods and it was unambiguously proved that the toxin infecting freshwater sh was mycolactone dia-F, a remote diastereomer of mycolactone F infecting saltwater sh.123 It should also be noted that mycolactone F-producing mycobacteria are not able to grow over 30 C, thereby limiting their virulence potential in humans.120 One role for these mycobacteria could be to act as an environmental reservoir for the evolution of human pathogens.120 The genome of the frog pathogen M. ulcerans ecovar Liandii 128FXT was completely sequenced in 2013 and is composed of 6.4 Mbp, 239 copies of IS2404, 4 copies of IS2606, 4994 CDs and 436 pseudogenes.112 The predicted genome of M. ulcerans ecovar Liandii and M. marinum DL240490 showed the existence of two plasmids, named pMUM002 (190 kb) and pMUM003 (210 kb) that differ from pMUM001 (Fig. 10).107 MLSA1 et A2 are identical to those from pMUM001 but these plasmids present differences in the mlsB gene encoding for the lower side chain which present a huge opportunity of recombination. As an example, the cyp140A7 gene is absent from pMUM002 and pMUM003 possibly due to the activity of IS2606 explaining the absence of an hydroxyl group at the position C100 of the mycolactones E and F.112,107 In addition, mycolactones E and F have a shorter lower side chain due to the deletion of module 4 in MLSB in pMUM002 and pMUM003. It should also be noted that a propionate unit is present on the loading module of the gene mlsB of pMUM002, thereby leading to the ethyl termination of the fatty acid side chain of mycolactone E. The inversion of the absolute congurations of the C110 - and C130 -hydroxyl groups of mycolactone F can be explained by important alterations in the MLSB modules 1 and 2 of pMUM003, as KRA were switched to KRB ketoreductases.82g,109 As a nal note on the genome of these two toxins, it was found that a high number of copies of IS2606 were present in the genome of mycolactone F but absent in the one of mycolactone dia-F, thus leading to the hypothesis that this insertion


NPR sequence might be responsible for an enhanced virulence or adaptation.101 6.4.5 Mycolactones S1 and S2. Compared to Africa or Australia, Japan has a limited record of proven Buruli ulcer cases.124 Since the rst report in 1982 in a 19-year old woman, only thirtytwo cases have been reported. This specic subspecies of M. ulcerans infecting Japan was termed shinshuense in 1989.125 From yellowish colonies of M. ulcerans subsp. shinshuense isolated in 2004 from a cutaneous lesion in a thirty seven-year old woman, Kishi isolated two new mycolactones named S1 and S2 in minute amounts, 0.6 and 0.4 mg from one culture dish.126 High-resolution mass spectroscopy, total synthesis (see ESI†) and comparison of chiral HPLC proles led to the structures shown in Fig. 9, that differs from mycolactone A/B by the presence of a ketone in C150 for mycolactone S1 and an additional (S)-hydroxyl group in C140 for mycolactone S2. 6.4.6 Mycolactone G. In addition to mycolactones A–F that have been isolated from different strains of M. ulcerans and M. marinum, a single genetically engineered mycolactone has been reported so far in the literature by Leadlay and collaborators.121 M. marinum DL045, a mycolactone F-producing mycobacteria isolated from a European sea bass cultured in the Mediterranean sea in Greece,118a was cloned with cyp140A7 (MUP_053), the accessory gene encoding for the P450 cytochrome cyp140A7, responsible for the post-PKS hydroxylation of the C120 position of the fatty acid side chain of mycolactone A/B (vide supra). The initial goal of these studies was to hydroxylate the C100 position of mycolactone F. The existence of a mycolactone with an additional oxygen atom was shown by LC-MS analyses and complementary analyses led to the structural determination of this new mycolactone which revealed that the oxidation had occurred on the C80 -methyl group and not at C100 as expected. This modication of the hydroxylation position was attributed by the authors to a minor modication of the orientation of the substrate into the active site of the enzyme. One of the most exciting aspects of M. ulcerans infections is the very rich biology that has spurred the interest of generations of researchers for the past 60 years. The most recent advances in this regard are discussed in the next section.

7 The biology of human M. ulcerans infections As discussed in Section 1, M. ulcerans infections in humans is due to the presence of mycolactone A/B, an unusual macrolidic toxin secreted by the mycobacteria. However, the exact mode of action of mycolactone A/B is still not accurately known, although it is of prime importance since it could be central to the development of new therapeutics and/or diagnosis tools. The infection leads to two main biological effects that were identied more than 30 years ago, namely a cytotoxic and an immunosuppressive effect.71,127,128 These effects have been studied in vitro on several types of cells (L929 murine broblasts, adipose cells,129 keratinocytes,130 cells of the inammatory system) and also in vivo in small animals and using footpad models. Major progress has been reported in the last 15 years

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concerning the vast biological proles of this toxin and the most signicant results are presented in the following sections.


7.1

Cytotoxic effects of mycolactone A/B

The cytopathic activities of M. ulcerans sterile ltrates and also of the puried toxin were proven in vitro on L929 murine broblasts and also in vivo on guinea pigs.71,73,74,108,111,127,131 Injection of mycolactone A/B led to the formation of ulcers (or related lesions) aer a few days, lesions that were identical to the ones observed on M. ulcerans infected patients. In addition, a cytoskeletal rearrangement of the L929 cells was observed using a wide range of mycolactone A/B concentrations, as low as 25 pg mL1 according to the initial 1999 report of Small et al.74, 20 nM being usually taken as the reference value. Rounding up of the cells (90 to 100%) is then observed aer 24 h followed by the detachment of the support and apoptosis (20–35%) aer 48 h.73,74,131 Recent detailed investigations using ow cytometry have estimated the concentration of mycolactone for which half of the cells were killed (LC50) and for which the metabolic activity of the cells was reduced by half (IC50). For mycolactone A/B, the LC50 was measured at 12 nM, the IC50 at 5 nM and the LC50/IC50 ratio at 2.4.111 It was also demonstrated that in the presence of either the acetone-soluble lipidic fraction of M. ulcerans or the isolated mycolactone A/B, the growth of L929 murine broblasts was stopped in the G0/G1 phase of the cell cycle.73,74,131 This effect seems to be reversible when the cells are no longer in contact with the toxin. Mycolactone A/B cytotoxicity was also proven by control experiments based on the use of a M. ulcerans mutant unable to biosynthesize mycolactone, named tox 1615A.74 In this case, no cell rounding was observed upon incubation with L929 broblasts and no ulcers were detected upon intradermal inoculation into guinea pigs, thereby strongly supporting the role of mycolactone A/B in virulence. The effect of mycolactone in patients seems to be dependent on the concentration of the toxin. Indeed, in the center of the lesion, necrosis with destruction of the adipose tissue is observed132 more than apoptosis. On the other hand, a lower concentration of the toxin leads to the predominance of apoptosis on the margins of the lesion. The lesions seem to be painless (see Section 2), which could be explained by toxin-mediated immunosuppression (see below) and also by nerve damage.31,133 Other effects can be observed on muscles; indeed injection of the toxin demonstrated a persistent loss in muscle force and also problems associated with growth and regeneration.134 Deciphering the mode of action of mycolactone A/B is a real challenge, which is further complicated by the fact the toxin is also able to diffuse from the infected tissues to the lymphoid organs via peripheral blood44,47,135 and can accumulate in internal organs with a preference for the lymphoid ones.44 Mycolactone was found intact in neutrophils, in mononuclear cells, in the draining lymph node (DLN)136 and in the spleen. A last point that ought to be mentioned in this section on naturally occurring mycolactone A/B is the absence of any antimicrobial activity on gram-positive (Streptococcus pneumoniae) as well as gram-negative (Neisseria meningitis, Escherichia

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coli) microbial species, as was shown in 2013.111 Furthermore, no activity was detected on yeast (Saccharomyces cerevisae) and amoeba (Dyctyostelium discoideum). This absence of activity is important to note since it contradicts the common belief that mycolactone A/B was an antimicrobial agent, preventing secondary bacterial infections of Buruli ulcer lesions. Actually, these secondary infections are much more common than anticipated and mycolactone A/B does not play any role in preventing them.57,111

7.2

The localization of mycolactone A/B in cells

The localization of the toxin in cells was studied by Small and coworker137 in 2003 and the Blanchard group138 in 2011 using uorescent derivatives of mycolactone A/B, obtained in a few steps from micrograms of the natural toxin extracted from M. ulcerans culture broth and from a diverted total synthesis approach, respectively (see Section 9.3 for details of the syntheses). The cytopathic activity on L929 cells of this uorescent compound was decreased by factor 6.5 compared to natural mycolactone A/B (130 nM vs. 20 nM, respectively). It was elegantly demonstrated that this modied mycolactone diffuses passively into the cell, and is exclusively localized in the cytoplasm of L929 murine broblasts, supporting a potential cytoplasmic target. Similar results were obtained from the incubation of a 14C-enriched mycolactone A/B with human epithelial cells and lymphocytes.80b To study the disruption of the actin cytoskeleton and its link with mycolactone A/B, the staining of actin was performed. The absence of actin staining/ uorescent mycolactone analogue colocalization led to the conclusion that mycolactone does not have a direct effect on the actin, a result that should be put into perspective with recent studies that found a clear link with a family of protein regulating the polymerization of actin (see Section 7.4 for a complete discussion).139

7.3

Mycolactone A/B-mediated immunosuppression

In 1988, the immunosuppressive effect induced by M. ulcerans infections was rst highlighted128 and the exact role of mycolactone A/B in this phenomenon has triggered numerous studies. Although the mechanism of immunosuppression is not clearly established, and in particular whether a local or a systemic immunosuppression is occurring,140 recent advances are presented in the following paragraphs. One of the rst cellular events seems to be the efficient phagocytosis of M. ulcerans by macrophages, followed by a progressive inhibition of phagocytic activity that correlates with toxin production, pointing towards the existence of an intramacrophage growth phase for the mycobacteria.141 The viability of dendritic cells is also dramatically impaired by mycolactone A/B. These key initiators and regulators of immune responses are no longer able to produce the chemotactics signals necessary to the immune response, even at non-cytotoxic concentrations.142 In addition, the migration of dendritic cells to the lymph node seems to be blocked and the recruitment of inammatory cells to the infection site is also limited. These


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Review results can explain the lack of an inammatory response, which correlates with the decrease of T lymphocyte activation. The primary immune response seems to be activated at the Buruli ulcer lesion, which was demonstrated by modication of mRNA levels for the various cytokines (TNF, IL-2, IL-10, IFN-g, etc.).143 Quite strikingly, a complete inhibition of tumour necrosis factor (TNF)144 produced by monocytes and macrophages following infection or incubation with mycolactone A/B has been observed.145 The biology of T cells is also profoundly modied by the action of the M. ulcerans toxin. In classical cases, a Type 1 helper T cell (Th1) adaptative immune response with an elevated production of interferon gamma (IFN-g, a macrophage-activating factor) is necessary to cope with a mycobacterial infection. However, in the specic case of Buruli ulcer infections, a modication of the Th1/Th2 balance is observed, Th2 being associated with the humoral immunity. When the disease is at the nodule stage, the Th1 response seems to be the predominant one, with an important production of IFN-g and IL-12, as well as a low level of IL-10.143c At the ulcerative stage, the production of TGF-b and IL-10 is important and a switch to the Th2 response is observed, associated with a diminution of the IFN-g level.146 We have to note here that the IFN-g response becomes normal again aer the excision of the lesion by surgery, indicating that the immunosuppressive effect is not the consequence of a genetical default in T cells but may be due to the presence of M. ulcerans.62d,147 The cytokines produced by the Th1 immune response inhibit the Th2 lymphocytes and vice-versa and the Th1/Th2 immunological balance also plays an important role in the M. ulcerans elimination. The Th1 response can be, for example, favored during a BCG vaccination.148 Overall, mycolactone A/B is an active immunosuppressor of the IL-2 and IFN-g production by T cells.80,121,135 Besides the mycolactone A/B-mediated inhibition of cytokine production from primary T cells, these toxins are also able to impair their migratory properties to peripheral lymph nodes (PLNs).149 This alteration of T-cell homing is also accompanied by a reduction of the expression of the L-selectin receptor (CD62-L). Another effect of mycolactone A/B is an hyperactivation of the Lck kinase of the Src family,145 via a relocalization in the microdomains of the plasma membrane, called lipids ras. This Lck kinase is expressed by the T lymphocytes and its activation leads to a signaling cascade. Mycolactone A/B triggers a decrease in the T cell receptor (TCR) expression level and an activation of PLC-g1, involving a depletion of the intracellular calcium level.150 These two mechanisms induced by the hyperactivation of Lck limits the responsiveness of T cells to stimulation and can contribute to apoptosis.

7.4 Mycolactone A/B-mimicking of endogenous regulators of WASP and N-WASP As discussed above, the biological effects of mycolactone A/B are numerous. It was for example noted that the associated toxicity proceeded via cytoskeletal rearrangement, detachment and


NPR eventually cell death. The link between mycolactone's effect and an actin-related event was therefore investigated and it was found in 2013 by Demangel and coworkers that mycolactone A/B was able to modulate Wiskott-Aldrich syndrome protein (WASP) and neural WASP (N-WASP), two members of a family of scaffold proteins that transduce a variety of signals into dynamic remodeling of the actin cytoskeleton.139,151 This result is clearly a landmark discovery since it is the rst target of mycolactone A/B to be published. In addition, it should be mentioned that mycolactone is the rst non-proteic molecule able to modulate WASP and N-WASP, thus highlighting once again the unique characteristics of this complex macrolide. Upon a suitable activation of N-terminal target sequences, a WASP and N-WASP C-terminal verprolin-colin-acidic (VCA) domain is released and interact with the ARP2/3 actin-nucleating complex, thereby stimulating actin polymerization. It was demonstrated that mycolactone A/B binds selectively to WASP and N-WASP, thus triggering a transition from an auto inhibited state to an active state with an efficient release of the VCA domain. It was also found that mycolactone A/B was a 100-fold more potent activator of N-WASP than CDC42, its major regulator in vivo. The main consequence of this hijack of WASP and N-WASP is an uncontrolled activation of the ARP2/3-mediated assembly of actin in the cytoplasm. In an exciting overlap of synthetic chemistry and immunology, uorescent mycolactone analogues were prepared by Blanchard and coworkers138 and used to explore colocalization of the toxins with active WASP (for details of the synthesis of these uorescent probes, see Section 9.2). A limited, but signicant colocalization was noted aer one hour. Considering also the increase in concentration of the N-WASP-mediated ARP2/3 complex in the perinuclear area of epithelial cells exposed to mycolactone, these results unambiguously demonstrated that mycolactone A/B promoted the recruitment and activation of ARP2/3 by N-WASP. The effect of mycolactone on the viability of epithelial cells was also investigated and quite intriguingly, it was found that the binding to N-WASP affected adhesion, speed of cell migration and direction sensing of epithelial cells. Epidermal thinning followed by N-WASP–mediated detachment and subsequent detachment-induced apoptosis (anoikis) resulted in rupture of the epiderm thus affording the rst mechanism of Buruli ulcer pathogenesis at a molecular level. As briey overviewed here, the fact that mycolactone A/B mimics endogenous regulators of WASP and N-WASP has far reaching implications that will certainly deeply inuence the directions of future research in chemistry and biology. As seen in this section, the biology of mycolactones A-S2 is very rich and has spurred a lot of interest since the discovery in 1948 of this “new mycobacterial infection in man”.1 Chemistry is also another area of research that has been very active since the report of mycolactone A/B in 1999 by Small and coworkers. The seducing structure of these toxins has elicited the interest of several research groups for structure conrmation and/or elucidation as well as illustration of synthetic methodologies. In the following section, the different strategies for the elaboration of mycolactone A/B will be detailed followed by a section on the

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synthesis of targeted analogues by Altmann,160 Kishi152 and Blanchard138 that have been central to the studies of the rst structure–activity relationships. In order to keep this review to a reasonable size, the total syntheses of mycolactones C, E, F, diaF, S1, S2 as well as the partial syntheses of mycolactone A/B are discussed in the Electronic Supplementary Information.†

8

Total syntheses of mycolactones A/B

The gross structure of mycolactone A/B was disclosed by Small and coworkers in 1999, a report that was soon followed by the complete assignment of the stereochemistry by Kishi thanks to his universal NMR database approach, rst of the C1–C20 fragment and then of the complete mycolactone A/B as will be developed in the next sub-sections. Following Kishi's lead,152 Negishi159 and Altmann160 published two other total syntheses of mycolactone A/B in 2011. An overview of the different

Chart 1

disconnections will be presented rst (Section 8.1), followed by the three generations of synthesis by Kishi (Section 8.2), and the total syntheses by Negishi and Altmann (Sections 8.3 and 8.4). The photochemical behaviour of mycolactone A/B by Kishi will then be discussed (Section 8.4) and will close this review of mycolactone A/B elaborations. 8.1 Overview of the synthetic strategies of Kishi, Negishi and Altmann The synthetic strategies of Kishi, Negishi and Altmann for the total synthesis of mycolactone A/B rely on a logical esterication reaction of the C5-hydroxyl function (Chart 1, eq. 1) leading to two fragments, the complex undecenolide C1–C20 1 and the southern fragment C10 –C160 2. In two strategies (Kishi and Altmann), the undecenolide 3 has been disconnected at the C13–C14 s bond thanks to a

Overview of the synthetic strategies for the total syntheses of mycolactone A/B by the groups of Kishi, Negishi and Altmann.

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Review palladium-catalyzed coupling reaction, leading to 4a or 9, while Negishi relied on a late stage macrolactonization of the secoacid 4b. The three C1–C13/C20 fragments 4a,b and 9 could be either in the open-chain form (4, eq. 2) or in the lactonic form (9, eq. 3). These fragments could be prepared via three different key reactions, namely a palladium-catalyzed cross-coupling reaction (eq. 2, Box 1), an epoxide opening (eq. 2, Box 2) or a ring-closing metathesis (eq. 3, Box 3). It should be noted here that this schematic representation of the synthetic strategies investigated by Kishi is quite simplied and takes into account only the second and third generation of total syntheses. On the other hand, only two disconnections have been adopted for the preparation of the C10 –C160 pentaenoic acid fragment (eq. 4): a Negishi cross-coupling for the construction of the C70 –C80 s bond (Box 4) and a Horner–Wadsworth– Emmons olenation for the C80 –C90 p bond (Box 5). The synthesis of mycolactone A/B will now be discussed from a chronological point of view, starting with the pioneering studies of Kishi.

8.2 Structural determination and total syntheses of mycolactone A/B by Kishi (2001–2010) 8.2.1 Relative and absolute conguration (2001). Following the determination of the gross structure of mycolactone A/B in 1999 by Small and collaborators,74,76 Kishi embarked in the total synthesis of the different stereoisomers of the C1–C20 fragment as well as of truncated derivatives of the southern fragment.153 The NMR analysis of these model compounds in achiral and

Scheme 1 Synthesis of four C13–C20 stereoclusters. Reagents and conditions: (1) TBSCl, imidazole, DMF; (2) DIBAL-H, CH2Cl2, 78 C; (3) Z-butene, tBuOK, n-BuLi, ()-(Ipc)2BOMe, BF3$OEt2, THF, 78 C then NaOH, H2O2, 1 h, 78%; (4) TBSCl, imidazole, DMF, 20 C, 93%; (5) O3, CH2Cl2, 78 C, PPh3, 99%; (6) Ph3P] C(Me)CO2Et, benzene, 60 C, 12 h, 77%; (7) DIBAL-H, CH2Cl2, 78 C, 77%; (8) TBAF, THF, 4 h, 80%.


NPR chiral solvents eventually led to the unambiguous determination of the relative and absolute conguration of these toxins. In order to establish the relative conguration of the seven stereocenters of the C1–C20 fragment, Kishi considered successively two clusters of this core fragment: the C13–C20 and then the C1–C20. Synthesis of the C13–C20 cluster starts from ethyl (3R)-()-methyl 3-hydroxybutyrate 15 (Scheme 1) thereby setting the conguration of the C19 stereocenter. Two trivial steps led to the corresponding b-silyloxyaldehyde that underwent an asymmetric crotylboration using (Z)-butene and ()-methoxydiisopinocampheylborane. Protection of the hydroxy group followed by ozonolysis of the terminal alkene and Wittig olenation led to the corresponding a,b-unsaturated ester. Reduction of the latter followed by global deprotection of the silylethers nally offered 16a. Implementation of this synthetic scheme using all possible combinations of (E)- and (Z)butene and (+)- or ()-methoxydiisopinocampheylborane nally gave the four possible diastereomers 16a–d. The 1H and 13C NMR of the latter were then recorded and compared with the data obtained from natural mycolactone A/B. The lowest chemical shi difference between 16a–d and mycolactone A/B, diagnostic of a similar structure, showed that the all-syn diastereomer 16a possessed the correct relative conguration at C16, C17 and C19. The synthesis of four representative diastereomeric C1–C20 fragments 28a–d (see Scheme 4) was then considered for NMR comparisons with mycolactone A/B, and for the sake of brevity, only the synthesis of diastereomer 28a (that turned out to be similar to the natural toxin) is detailed here. This challenging

Scheme 2 Synthesis of the C1–C7 (eq. 1) and C8–C17 (eq. 2) fragments. Reagents and conditions: (1) Z-butene, tBuOK, n-BuLi, (+)-(Ipc)2BOMe, BF3$OEt2, THF, 78 C then NaOH, H2O2, 1 h, 80%; (2) TBSCl, imidazole, DMF, 96%; (3) O3, CH2Cl2, 78 C, PPh3; (4) NaBH4, EtOH, 82% (2 steps); (5) Ph3P, I2, CH2Cl2, 88%; (6) m-CPBA, CH2Cl2, 0 to 20 C, 80%; (7) propyne, THF, n-BuLi, BF3$OEt2, 78 C, 94%; (8) TBAF, THF, 73%; (9) cyclopentanone, TsOH, benzene, 76%; (10) Cp2ZrHCl, THF, 50 C, 1 h; (11) I2, THF, 62%.

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Scheme 3 Synthesis of the C1–C13 and C14–C20 fragments. Reagents and conditions: (1) 1. tBuLi, THF, 78 C then ZnCl2, 78 to 20 C; (2) 21, Pd(PPh3)4 (10 mol%), 60%; (3) TFA, CH2Cl2, 77%; (4) PivCl, Pyridine, 99%; (5) TESCl, CH2Cl2, Imidazole, 91%; (6) DIBAL-H, CH2Cl2, 78 C, 98%; (7) PPh3, I2, Et2O/MeCN, 91%; (8) TsOH, MeOH/THF, 79%; (9) Cyclopentanone, TsOH, 83%; (10) O3, CH2Cl2 then PPh3, 78 C, 97%; (11) DAMP, tBuOK, THF, 78 C, 88%; (12) n-BuLi, MeI, 78 to 20 C, 99%; (13) Cp2ZrHCl, THF, 50 C then I2, THF, 79%.

endeavor relies on two Negishi couplings for the construction of the C7–C8 and C13–C14 s bonds. The rst coupling requires the synthesis of the C1–C7 and C8–C13 fragments (Scheme 2, eqs. 1 and 2). The two stereocenters at C5 and C6 were set up via an asymmetric crotylboration of aldehyde 17 followed by protection of the hydroxyl function and transformation of the terminal alkene in the primary iodide 18 in three steps. Meanwhile, the C8–C13 vinyliodide 21 was prepared from methyl (S)(+)-3-hydroxy-2-methylpropionate through the epoxidation of 19 (diastereomeric ratio not determined) followed by ring opening with lithiopropyne, protection of the 1,3-diol as an acetal and hydrozirconation/iodolysis of the alkyne. Coupling of these two subunits under Negishi's conditions led to the fragment 22 in good yield (Scheme 3, eq. 1). Further functional groups manipulations offered the C1–C13 derivative 23 ready for the second Negishi coupling with the C14–C20 fragment. The latter was prepared in six steps from the previously prepared 24, by ozonolysis of the terminal alkene followed by installation of the methylated alkyne via a Seyferth–Gilbert reaction (Scheme 3, eq. 2). A last hydrozirconation/iodolysis nally led to the trisubstituted vinyl iodide 26.

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Scheme 4 Completion of the C1–C20 fragment synthesis. Reagents and conditions: (1) tBuLi, THF, ZnCl2, 78 to 20 C then 26, Pd(PPh3)4 (10 mol%), 50%; (2) HF$pyr, pyridine, THF, 72%; (3) TEMPO, NCS, CH2Cl2, 95%; (4) NaClO2, NaH2PO4, 1,3-dimethoxybenzene, DMSO/tBuOH, 94%; (5) Cl3C6H2COCl, i-Pr2NEt, DMAP, benzene, 70%; (6) TFA, CH2Cl2, H2O, 62%; (7) HF$pyr, pyridine, THF, 77%.

Negishi coupling of the C1–C13 and C14–C20 fragments led to an advanced intermediate that was further transformed in the seco-acid 27 in three classical steps (Scheme 4). Macrolactonization under Yamaguchi's condition offered the desired undecenolide in good yield and global deprotection delivered the macrolactonic triol 28a. Implementation of this synthetic strategy allowed the preparation of the three complementary diastereomers 28b–d. NMR analysis of these fragments with the C1–C20 fragment of natural mycolactone A/B (prepared by saponication of the C10 –C160 southern fragment) conrmed that the relative conguration of the C5, C6, C11 and C12 stereocenters of the natural mycolactone corresponds to diastereomer 28a. In order to prove the relationship between the C5–C12 and the C16–C19 stereoclusters, the (C16,C17,C19)-epi-28a derivative was prepared and the 1H NMR of the tri-(S)- and tri-(R)-Mosher esters of 28a, (C16,C17,C19)-epi-28a and of the C1–C20 fragment of natural mycolactone were compared, thus proving that the relative and absolute congurations of the C1–C20 portion of mycolactones corresponds to diastereomer 28a. Using his concept of universal NMR database in chiral solvents, Kishi then addressed the stereostructure of the southern C10 –C160 fragment of mycolactone A/B. 154 Four This journal is ª The Royal Society of Chemistry 2013

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Scheme 5 Synthesis of model compounds of the C70 –C160 fragment. Reagents and conditions: (1) Roush tartrate ester modified allylboronate, toluene, 78 C; (2) NaH, BnBr, DMF, 88%; (3) OsO4, NMO, DABCO, THF/H2O; (4) Pb(OAc)4, benzene; (5) Me2CuLi, Et2O, 25 C, 83% (3 steps), 2 epimers (1 : 1); (6) Pd/C, H2, EtOH, 34%; (7) NaH, BnBr, TBAI, DMF, 66%; (8) 75% aq. AcOH; (9) 3,4˚ MS, CH2Cl2, 59% (2 steps); (10) DIBAL-H, Et2O, 90%; (MeO)2C6H4CHO, H2SO4, 3 A (11) SO3$py, i-Pr2NEt, DMSO, CH2Cl2; (12) Ph3P¼C(Me)CO2Et, THF, 80% (2 steps); (13) BCl3, CH2Cl2, 64%; (14) TBSOTf, 2,6-lutidine, CH2Cl2, 95%; (15) DIBAL-H, CH2Cl2, 69%; (16) SO3$py, i-Pr2NEt, DMSO, CH2Cl2; (17) (MeO)2P(O)CH2CO2Me, NaH, benzene, 71% (2 steps); (18) TBAF, THF, 80%.

diastereomers of the model compound were prepared from Dglyceraldehyde acetonide 29 (Scheme 5). Roush allylboration of the latter led to homoallylic alcohol 30 that was further transformed to an intermediate aldehyde. A non-selective methylcupration followed by benzyl ether deprotection delivered diol 31 as a mixture of separable diastereomers, the conguration of which being determined by 13C NMR analysis of the corresponding diacetonides and also chemical correlation from L-arabinose (not shown). Protecting group manipulation then delivered the primary alcohol 32 whose C120 and C130 ,C150 hydroxy groups were differentially protected. Oxidation of 32 followed by Wittig olenation and global deprotection afforded the trihydroxy a,b-unsaturated ester 33. The latter was nally extended to the corresponding a,b,g,d-unsaturated ester 34a. Implementation of this synthetic strategy allowed the preparation of the three complementary diastereomers 34b–d. Comparison of the 13C and 1H NMR of the four diastereomers with the corresponding portion of natural mycolactone A/B demonstrated the all-syn relationship of the C120 ,C130 ,C150 stereocenters. Further 1H NMR analysis of 34a–d and of natural


NPR mycolactone A/B in chiral solvents ((R)- and (S)-N,a-dimethylbenzylamine) unambiguously established the absolute conguration of the C120 ,C130 ,C150 stereocenters and by extension of the complete mycolactone A/B. This very elegant and insightful work by Kishi showcases the power of the combination of stereoselective synthesis of model compounds and their NMR analysis in achiral and chiral solvents. Decades aer the rst description of Buruli ulcer, the exact structure of the virulence factor, mycolactone A/B, was nally established. 8.2.2 First generation total synthesis (2002). A year later, Kishi reported the rst total synthesis of mycolactone A/B based on esterication of the C5-hydroxy group of the C1–C20 fragment with the pentaenoic acid.155 Compared to the 2001 studies that established the absolute conguration of the fatty acid side chain, an efficient synthesis of the latter was developed using a Horner–Wadsworth–Emmons olenation as a key step (Scheme 6).156 Phosphonate 38 was prepared from allyl alcohol using a sequence of reduction, oxidation and Wittig olenation leading to the corresponding ethyl dienoate 37 (eq. 1). Repetition of this reduction, oxidation and Wittig olenation sequence was then followed by uoride-mediated deprotection of the primary silyl ether and conversion of the latter to the corresponding bromide. An Arbuzov reaction nally delivered the targeted phosphonate 38, the rst partner of the Horner– Wadsworth–Emmons olenation. The second partner was prepared from b-silyloxy aldehyde 39, which was rst

Scheme 6 Synthesis of the C10 –C80 (eq. 1) and C90 –C160 (eq. 2) fragments. Reagents and conditions: (1) TBSCl, imidazole, DMF; (2) O3, CH2Cl2, 78 C; (3) Ph3P]C(Me)CO2Et, CH2Cl2; (4) DIBAL-H, CH2Cl2, 78 C; (5) SO3$py, i-Pr2NEt, CH2Cl2/DMSO (3 : 2); (6) Ph3P]C(Me)CO2Et, benzene, 90 C, 20% (6 steps); (7) repeat steps 4 to 6, 89% (3 steps); (8) TBAF, THF, 87%; (9) PBr3, Et2O, 77%; (10) (EtO)3P, 90 C, 96%; (11) NaH, (EtO)2P(O)CH2COOEt, benzene, 64%; (12) AD-mix a, MeSO2NH2, tBuOH/H2O (1 : 1), 40 h, 0 C, 70%; (13) TBSOTf, 2,6-lutidine, CH2Cl2, 0 C, 99%; (14) DIBAL-H, CH2Cl2, 89%; (15) SO3$py, i-Pr2NEt, CH2Cl2/ DMSO (3 : 2); (16) Ph3P]C(Me)CO2Et, toluene, 110 C, 83% (2 steps); (17) DIBALH, CH2Cl2, 78 C, 57%; (18) SO3$py, i-Pr2NEt, CH2Cl2/DMSO (3 : 2), 100%.

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Scheme 7 Synthesis of the C10 –C160 fragment. Reagents and conditions: (1) LDA, 42, THF, 78 to 0 C, 94%; (2) tungsten lamp, acetone-d6; (3) LiOH, THF/ MeOH/H2O, 20 C, 100%.

transformed into the corresponding a,b-unsaturated ethyl ester and then submitted to an asymmetric dihydroxylation reaction leading to 40 (eq. 2). A moderate diastereoselectivity was observed (3.8 : 1), which contrasts with the closely related chemo- and diastereoselective dihydroxylation of the corresponding dienoate that was developed in the diverted total syntheses of mycolactone analogues (see Section 9.2).138 Further functional group transformations led in six steps to the a,bunsaturated aldehyde 42. Deprotonation of 38 with lithium diisopropylamide and condensation of the corresponding anion to aldehyde 42 led to the C10 –C160 fragment as a mixture of geometrical isomers, the structures of which being deduced from 1H NMR analysis (Scheme 7). Photochemical equilibration of this mixture using a tungsten lamp or even under irradiation of classical laboratory lights led to the following ratio: (20 E,40 E,60 E,80 E,100 E)-43/(20 E,40 Z,60 E,80 E,100 E)43/(20 E,40 E,60 Z,80 E,100 E)-43/(20 E,40 Z,60 Z,80 E,100 E)-43 ¼ 36 : 52 : 4 : 5, plus 3% of two minor isomers. Finally, saponication of the methyl ester with lithine delivered the carboxylic acid 44 in quantitative 0 0 0 0 yield. The Z-D4 ,5 /E-D4 ,5 ¼ 60 : 40 ratio of 44 is a simplication of the six isomers ratio presented above, taking into account only the two major isomers. Having secured access to the C10 –C160 fragment, attention was then focused on the esterication of the C5-hydroxy function. First, the C17,C19-diol of compound 28a was selectively protected as a cyclopentylidene acetal with 1,1-dimethoxycyclopentane under acidic conditions (Scheme 8, eq. 1). The esterication itself was then considered (eq. 2). Classical conditions such as N-(3-dimethylaminopropyl)-N0 -ethylcarbodiimide hydrochloride (EDCI) or (benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexauorophosphate (BOP)

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Scheme 8 Completion of the total synthesis of mycolactone A/B. Reagents and conditions: (1) 1,1-dimethoxycyclopentane, p-TsOH, benzene, 20 C; (2) 45, Cl3C6H2COCl, i-Pr2NEt, DMAP, benzene, 20 C, 90%; (3) TBAF, THF, 20 C, 81%; (4) AcOH/H2O/THF, 20 C, 67% after one recycling.

combined with 4-dimethylaminopyridine being unsuccessful, Yamaguchi conditions were employed leading to the desired protected mycolactone A/B as a 3 : 2 mixture of 40 Z and 40 E isomers in 90% yield. Global unmasking of protected complex natural products is known to be an extremely challenging task owing to delicate structural arrangements. In addition, the scale of the deprotection reaction is oen an important parameter. The total synthesis of mycolactone A/B is no exception, as we will see in this review since several deprotection strategies were adopted in the past ten years for this very last step. With regard to the 2002 total synthesis of Kishi, a global deprotection of the three silyl ethers and of the cyclopentylidene acetal with HF$pyridine in acetonitrile led to mycolactone A/B in only 5–10% yield. A twostep sequence was then adopted using tetrabutylammonium uoride (TBAF) followed by two successive treatments with an aqueous acetic acid solution. As noted by Kishi, side-product formation was not avoided but only minimized, delivering synthetic mycolactone A/B in 67% yield. To exclude the possibility that this synthetic material could be remote diastereomers of natural mycolactone A/B, NMR analysis were performed in chiral solvents such as (R)- and (S)-DMBAs (see Section 8.2.1). It was found that the Dd proles of the synthetic and natural toxins were identical, thus conrming the proposed structures. In addition, biological


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evaluation of the synthetic and natural toxins led to similar results. This rst generation total synthesis is a remarkable achievement that was further rened in 2007, taking into account some protecting groups issues as will be discussed in the next sub-section. 8.2.3 Second generation total synthesis (2007). Analysis of the rst generation total synthesis revealed unanticipated issues with the C17,C19-cyclopentylidene acetal deprotection. Wishing to develop a more straightforward synthesis of mycolactone A/B, Kishi reported a second generation of total synthesis in 2007 that relied on a single set of silyl ethers for the protection of the C17, C19, C120 , C130 and C150 hydroxyl groups. In addition, the strategic ordering of C13–C14 s bond creation and macrolactonization is reversed for the C1–C20 fragment compared to the rst generation total synthesis.157 From a synthetic perspective, the elaboration of the C1–C7 and C14–C20 fragments 48 and 49 is similar to the rst

generation, the only differences being the nature of the protecting groups at C5, C17 and C19 and the oxidation state of the C1 carbon (Scheme 9, eqs. 1 and 2). On the other hand, the synthesis of the C8–C13 fragment 21 was totally revised to avoid the unselective epoxidation reaction creating the C11 stereocenter (compare Scheme 9, eq. 3 and Scheme 2). The lithium enolate of the (S)-diethyl malate 50 was alkylated with methyl iodide following Seebach's procedure, affording the corresponding hydroxy diester as an anti/syn mixture of 8 : 1. Aer reduction and selective protection of the primary alcohol, diol 51 (d.r. ¼ 8 : 1) was obtained in good yield. Conversion of the latter to the terminal epoxide followed by ring opening with lithiopropyne delivered the homopropargylic alcohol 52. A two-step sequence then led to the corresponding 8 : 1 diastereomeric cyclopentylidene acetals that could be separated by ash chromatography. Hydrozirconation and iodolysis delivered the desired trisubstituted vinyliodide 21 in good yield.

Scheme 9 Syntheses of the C1–C7 (eq. 1), C14–C20 (eq. 2) and C8–C13 (eq. 3) fragments. Reagents and conditions: (1) Z-butene, tBuOK, n-BuLi, (+)-(Ipc)2BOMe, BF3$OEt2, THF, 78 C, 80%; (2) NaH, PMBBr, DMF, 89%; (3) TBAF, THF, 96%; (4) SO3$py, i-Pr2NEt2, CH2Cl2/DMSO, 94%; (5) NaClO2, NaH2PO4, 2-methyl-2-butene, t BuOH/H2O; (6) MeI, DBU, CH3CN, 84% (2 steps); (7) OsO4, NMO, acetone/H2O, 80%; (8) Pb(OAc)4, benzene; (9) NaBH4, MeOH, 83% (2 steps); (10) Ph3P, I2, CH2Cl2, 92%; (11) TBSCl, imidazole, DMF, 100%; (12) O3, CH2Cl2, 94%; (13) DAMP, tBuOK, THF, 78 C, 84%; (14) n-BuLi, MeI, 93%; (15) Cp2ZrHCl, THF, 50 C then I2, 65%; (16) LDA, MeI, THF, 78 C, 80%, d.r. ¼ 8 : 1; (17) LiAlH4, THF, reflux; (18) n-Bu2SnO, MeOH, reflux; (19) TBSCl, CHCl3, 70%, (3 steps); (20) Ts-Imidazole, NaH, THF, 88%; (21) n-BuLi, propyne, BF3$OEt2, THF, 78 C, 96%; (22) TBAF, THF, 96%; (23) cyclopentanone, p-TsOH, benzene, 76%; (24) Cp2ZrHCl, THF, 50 C then I2, 62%.

Scheme 10 Completion of the second generation of total synthesis of mycolactone A/B. Reagents and conditions: (1) 21, Zn, Cu(OAc)2, Pd(Ph3P)4, LiCl, NMP, 60 C, 83%; (2) TFA, CH2Cl2, H2O, 90%; (3) TIPSCl, imidazole, DMF, 100%; (4) LiOH, THF/MeOH/H2O, 81%; (5) Cl3C6H2COCl, i-Pr2NEt, DMAP, benzene, 96%; (6) HF$pyridine, pyridine, CH3CN, 90%; (7) Ph3P, I2, imidazole, CH2Cl2, 98%; (8) Zn, Cu(OAc)2; (9) 49, Pd(Ph3P)4 (15 mol%), LiCl, NMP, 60 C, 80%; (10) DDQ, CH2Cl2/ H2O, 91%; (11) 44 (see Scheme 7), Cl3C6H2COCl, i-Pr2NEt, DMAP, benzene, 20 C; (12) TBAF, THF, 72% (2 steps).


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NPR Having in hand the three building blocks 21, 48 and 49, the Negishi couplings planned for the creation of the C7–C8 and C13–C14 s bonds were studied (Scheme 10). Owing to the oxidation state of C1 in 48, a modication of the Negishi coupling was required since the classical lithium to zinc transmetalation would not be compatible with the C1-ester motif. Kishi found that insertion of an active zinc-copper couple into the C7-iodide s bond of 48 was very efficient and led to a high yield of the coupled product using modied Corey's catalytic system, palladium(0) and lithium chloride in a polar aprotic solvent. This advanced intermediate was then transformed into seco-acid 53 in three steps and was further cyclized to the desired undecenolide under Yamaguchi's conditions. Deprotection of the C13-silylether and conversion into the corresponding primary alkyliodide 54 occurred smoothly, opening the way for the second key Negishi cross-coupling. Compound 54 was transformed into the corresponding alkylzinc iodide using zinc-copper couple and then cross-coupled with vinyliodide 49, affording the complete C1–C20 fragment 55 in excellent yield aer selective deprotection of the C5-para methoxybenzylether. Completion of the synthesis called for the Yamaguchi esterication of the C1–C20 fragment with the fatty acid side chain 44 (prepared in Scheme 7) followed by global deprotection using tetrabutylammonium uoride in 72% yield for two steps. This second generation total synthesis implemented major improvements over the rst generation, improving the selectivity and efficiency of the synthetic strategy. However, Kishi developed a third generation total synthesis with the specic aim of shortening the synthesis as well as achieving a scalable total synthesis of the C1–C20 fragment of mycolactone. These efforts are detailed in the following sub-section. 8.2.4 Third generation total synthesis (2010). As in the second generation of total synthesis, the C1–C20 fragment synthetic strategy encompassed a redox economy for the C1carbon that is at the carboxylic acid oxidation state from the beginning (Scheme 11, eq. 1).158 Starting from aldehydo-ester 17, an asymmetric syn-selective crotylboration delivered the corresponding hydroxyester in 86% ee, without competitive formation of the corresponding d-lactone. Protection of the hydroxyl group as a para-methoxybenzylether required an extensive optimization as the d-lactonic product was obtained under classical conditions. It was eventually found that scandium(III) triate was a convenient catalyst for this transformation, delivering ester 47 in good yield. Further functional group manipulations led to the u-iodoester 48 (86% ee) in four steps. The synthesis of the C8–C13 fragment was also completely revised (Scheme 11, eq. 2) since the rst and second generation total syntheses were plagued with moderate selectivities for the control of the C11- and C12-stereocenters, respectively (see Schemes 2 and 9). Starting from the known 1,4diyne 56, a chemo- and stereoselective hydroalumination delivered compound 57 that was epoxidized using Sharpless conditions, leading to epoxyalcohol 58 in 80% ee that could be recrystallized to 99% ee and 70% yield. Ring-opening by a higher-order cuprate followed by acetonide formation offered the alkyne 20 that was transformed in the corresponding

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Review

Scheme 11 Syntheses of the C1–C7 (eq. 1) and C8–C13 (eq. 2) fragments. Reagents and conditions: (1) Z-butene, tBuOK, n-BuLi, (+)-(Ipc)2BOMe, BF3$OEt2, THF, 78 C, 78%; (2) PMB-trichloroacetimidate, Sc(OTf)3 (5 mol%), toluene, 0 C, 81%; (3) OsO4 (2 mol%), NMO, acetone/H2O; (4) Pb(OAc)4, benzene; (5) NaBH4, MeOH, 0 C, 71% (3 steps); (6) Ph3P, I2, imidazole, CH2Cl2, 92%; (7) LiAlH4, Et2O, ˚ CH2Cl2, 98%; (8) Ti(O-i-Pr)4 (10 mol%); ()-DET (8 mol%), tBuOOH, MS 4 A, 25 C, 70%; (9) MeLi, CuCN, THF, 20 C, 79%; (10) p-TsOH, cyclopentanone, 89%; (11) Cp2ZrCl2, Red-Al, THF, 50 C; (12) I2, THF, 68%.

trisubstituted vinyliodide with an excellent regioisomeric ratio (21/59 ¼ 22 : 1) and in enantiomerically pure form. It is worth noting that the mode of preparation of the Schwartz reagent had a direct impact on the regioselectivity as well as on the level of side-products, a combination of zirconocene dichloride and sodium bis(2-methoxyethoxy)aluminum dihydride (Red-Al) giving the best results according to Kishi. Compared to the rst and second generations syntheses, elaboration of the C14–C20 fragment relied on catalytic asymmetric reactions to set up the C16- and C17-stereocenters (Scheme 12, eq. 1). The readily available (R)-propylene oxide 60 was converted into 1,3-enyne 63 in ve steps, thus setting the stage for a titanium-catalyzed asymmetric epoxidation reaction using Katsuki's ligand 65. An impressive diastereoselectivity, superior to 50 : 1, was obtained. A regioselective and stereospecic epoxide opening using a methyl alanate delivered the corresponding alcohol in excellent yield and protection followed by Schwartz hydrozirconation/iodolysis nally led to the C14–C20 northern fragment 49.


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NPR


Scalable syntheses of the three key fragments in hand, Kishi proceeded to the union of 21, 48 and 49 using the Negishi crosscoupling developed in the second generation total synthesis (Scheme 12, eq. 2). An excellent yield was obtained for the C7– C8 s bond construction between 21 and 48 and aer acetal cleavage, one of the two minor diastereomers could be chromatographically removed. Protection of the C13-primary alcohol followed by saponication of the methyl ester delivered the seco-acid 53. Yamaguchi macrolactonization led to the desired undecenolide in enantiomerically pure form, and the last minor diastereomer could be conveniently removed by chromatography. A classical two-step procedure nally led to the C13-primary iodide, precursor of the last Negishi coupling. Using the reported conditions, the latter delivered the fully protected macrolactone in excellent yield. It should be emphasized here that this strategy allowed the preparation of six grams of macrolactone 55a, an astonishing achievement in the eld of natural products syntheses, highlighting once again the efficiency of Kishi's third generation of the C1–C20 portion of mycolactone. Besides these seminal total syntheses of mycolactone A/B, Kishi reported the total syntheses of mycolactones C, E, F, dia-F, S1 and S2. In order to keep this synthetic section focused on mycolactone A/B, these elegant achievements are not discussed and are presented in the ESI.† Coming back to mycolactone A/B, Negishi and Altmann have reported two other elaborations of these complex toxins in 2011, which were preceded by partial syntheses disclosed in 2006 and 2007, respectively. These accomplishments are detailed in the following two sections.

8.3

Scheme 12 Completion of the third generation of total syntheses of mycolactone A/B. Reagents and conditions: (1) n-BuLi, ethynyltrimethylsilane, BF3$OEt2, Et2O, 78 C; (2) TBSCl, imidazole, DMF; (3) NIS, AgNO3, DMF; (4) Cy2BH, THF, 0 C, then AcOH, 95% (4 steps); (5) propyne, PdCl2(PPh3)2 (4 mol%), CuI (8 mol%), Et2NH, 20 C, 96%; (6) Ti(OiPr)4 (10 mol%), 65 (11 mol%), 4,40 -thiobis(6-tbutyl-mcresol), H2O2, phosphate buffer, CH2Cl2, 40 C, 91%; (7) LiAlMe4, BF3$OEt2, CH2Cl2, 78 C, 87%; (8) TBSCl, imidazole, DMF, 20 C, 99%; (9) Cp2Zr(H)Cl, THF, 50 C; (10) I2, THF, 0 C, 68%; (11) Zn, Cu(OAc)2; (12) 21 (as a 22 : 1 mixture of regioisomers), Pd(Ph3P)4 (10 mol%), LiCl (8.4 equiv.), NMP, 55 C, 95%; (13) TFA/ H2O/CH2Cl2, 91%; (14) TIPSOTf, 2,6-lutidine, CH2Cl2, 78 C; (15) LiOH, H2O/ MeOH/THF, 88% (2 steps); (16) Cl3C6H2COCl, i-Pr2NEt, DMAP, benzene, 74%; (17) HF$pyridine, pyridine, CH3CN, 92%; (18) Ph3P, I2, imidazole, CH2Cl2, 98%; (19) Zn, Cu(OAc)2; (20) 49, Pd(Ph3P)4 (15 mol%), LiCl (6 equiv.), NMP, 60 C; (21) DDQ, CH2Cl2/H2O, 91%.


Total synthesis of mycolactone A/B by Negishi (2011)

An elegant total synthesis of mycolactone A/B was reported in 2011 by Negishi, featuring several stereoselective palladiumcatalyzed and aluminum-mediated carbon-carbon bonds formation as key steps.159 As presented earlier in Chart 1, the bond disconnections in Negishi's approach are clearly different from Kishi's, except for the esterication of the C5-hydroxyl group. The synthesis of the C1–C20 portion of mycolactone A/B will be discussed rst, followed by the stereoselective elaboration of the southern C10 –C160 -fragment (Scheme 13). From a retrosynthetic point of view, the C1–C20 unit of mycolactone A/B was obtained via the epoxide-opening of compound 7 (corresponding to the C10–C20 fragment, see Chart 1, Box 2, Section 8.1) by an alkenylaluminate reagent 8. The rst fragment that we shall consider here is the C1–C9 subunit (Scheme 13, eq. 1). The latter was prepared through a conventional route starting from homoallylic alcohol 68, itself obtained from 1,5-pentanediol 67. Protection of the hydroxyl group of 68 as a silylether followed by hydroboration delivered the primary alcohol 69 in good yield. Compound 69 was then transformed into the terminal alkyne 70 using an oxidation reaction followed by formation of the dibromoolen and elimination according to the Corey–Fuchs procedure. Synthesis of the second reaction partner, terminal epoxide 76, starts from the known intermediate 71 already reported by Kishi

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Review

Scheme 14 Forging of the C9–C10 s bond en route to the C1–C20 fragment. Reagents and conditions: (1) AlMe3, ZrCp2Cl2 (7 mol%), CH2Cl2, 40 C; (2) n-BuLi, 78 C; (3) 76, 43 to 20 C, 83%; (4) TBAF, THF, 0 C, 78%; (5) TEMPO (4 mol%), PhI(OAc)2, CH2Cl2/H2O, 20 C; (6) NaClO2, NaH2PO4, tBuOH/H2O/2-methyl-2butene, 66%; (7) Cl3C6H2COCl, DMAP, i-Pr2NEt, benzene, 20 C, 78%; (8) HF$pyridine, THF, 20 C, 67%; (9) 1,1-dimethoxycyclopentane, p-TsOH (20 mol%), benzene, 20 C, 80%.

Scheme 13 Syntheses of the C1–C9 (eq. 1) and C10–C20 (eq. 2) fragments. Reagents and conditions: (1) TBSCl, imidazole; (2) (COCl)2, DMSO, Et3N, 75%; (3) Z-butene, tBuOK, n-BuLi, (+)-(Ipc)2BOMe, BF3$OEt2, THF, 78 C then NaOH, H2O2, 84%; (4) TBSOTf, lutidine; (5) BH3$THF, THF then H2O2, NaOH, 74%; (6) (COCl)2, DMSO, Et3N, CH2Cl2, 78 to 20 C; (7) PPh3, CBr4, Zn, CH2Cl2, 0 to 20 C; (8) n-BuLi, THF, 78 C, 74% (3 steps); (9) OsO4 (1 mol%), NMO, THF/H2O; (10) NaIO4, THF/ H2O, 94%; (11) s-BuLi, 77, THF, 78 to 20 C; (12) CF3COOH, THF, 0 C, 91%; (13) NaBH4, MeOH, 0 C; (14) CBr4, PPh3, 2,6-lutidine, 91% (2 steps); (15) 78, Pd2(dba)3 (2.5 mol%), tris(o-furyl)phosphine (10 mol%), DMF, 20 C, 89%; (16) MgBr2$Et2O, Et2O, 20 C; (17) Ti(OiPr)4 (100 mol%), ()-DIPT (100 mol%), tBuOOH, CH2Cl2, 78 to 23 C, 76% (2 steps); (18) LiBH4, BF3$OEt2, CH2Cl2, 40 C, 75%; (19) MsCl, 2,4,6-collidine, CH2Cl2, 0 C; (20) K2CO3, MeOH, 87% (2 steps).

(Scheme 13, eq. 2). Conversion of 71 to the corresponding aldehyde followed by a Corey–Schlessinger–Mills-modied Peterson-olenation using the lithium anion of 77 delivered the a,b-unsaturated aldehyde 72 in excellent yield. A two-step sequence then led to the allylic bromide 73 that underwent a palladium-catalyzed cross-coupling reaction with the vinylzinc bromide 78 in 89% yield. The perfect stereocontrol of this carbon–carbon s bond formation originated from the d-methyl branched substitution of the g-monosubstituted allylic bromide

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73. In a subsequent step, C10-deprotection delivered the allylic alcohol 74 that was submitted to a Sharpless epoxidation. Epoxyalcohol 75 was obtained as a single diastereo- and enantiomer that was further processed to the terminal epoxide 76 in good yield. Inspired by Negishi's own work in 1980, compound 70 was treated with trimethylaluminum in the presence of a catalytic amount of zirconocene dichloride (Scheme 14). The resulting vinylalane was then converted to the ate-complex using butyllithium, thus allowing a smooth opening of the epoxide 76 in 83% yield. Cleavage of the primary silylether of 79 followed by oxidation to the carboxylic acid 80, Yamaguchi macrolactonization and global deprotection led to 28a, a unique stereoisomer of the C1–C20 fragment of mycolactone A/B. Finally, selective protection of the C17,C19-diol motif of 28a was accomplished through the formation of the corresponding acetal 45, leaving the C5-hydroxy group unmasked for the esterication reaction with the C10 –C160 -southern fragment of mycolactone. The attention was then focused on the synthesis of the southern C10 –C160 -fragment 44. Negishi developed perfect stereoselectivities in the palladium-catalyzed carbon-carbon bond formation in the mid 19700 s. This total synthesis was therefore a unique opportunity to develop the rst access to a stereochemically homogeneous (40 Z)- or (40 E)-44, precursor of mycolactone A or B respectively, as was presented in the overview of the different synthetic blueprints (see Chart 1, Box 4, Section 8.1). The implementation of this strategy calls for the synthesis of the stereochemically pure (40 Z)- or (40 E)-C10 –C70 fragments as well as C80 –C160 fragment. This journal is ª The Royal Society of Chemistry 2013

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NPR the dienyne (40 Z)-83 as a single stereoisomer in good yield aer uoride deprotection. The second strategy (eq. 2)159b is much shorter and featured the bromoboration of propyne 84 followed by two consecutive palladium-catalyzed Negishi couplings, leading to dienyne (40 Z)-83 as a unique isomer. A nal zirconium-catalyzed methylalumination/iodolysis of the terminal alkyne of (40 Z)-83 and protection of the primary alcohol gave the C10 –C70 portion of mycolactone A, (40 Z)-86. On the other hand, the C10 –C70 -fragment of mycolactone B was prepared from propargylic alcohol 81 (Scheme 15, eq. 3). A rst zirconium-mediated hydroalumination/iodolysis of the alkyne followed by palladium-catalyzed cross-coupling with diethynylzinc afforded 2-penten-4-yn-1-ol 90. Zirconium-catalyzed methyl-alumination and iodolysis followed by a second palladium-catalyzed cross-coupling with diethynylzinc afforded dienyne (40 E)-83 as a unique stereoisomer. A last sequence of zirconium-catalyzed methyl-alumination, iodolysis and silylether formation delivered (40 E)-86, the precursor of the fatty acid side chain of mycolactone B as a single stereoisomer. Synthesis of the C80 –C160 fragment 93 started from commercially available (S)-ethyl 3-hydroxybutyrate ent-15 (Scheme 16). Aer protection of the hydroxyl group as a silylether and reduction of the ester to the aldehyde, an asymmetric syn-selective crotylboration using 94 was performed that afforded alcohol 91 as a unique isomer aer protection. The double bond of the latter was then dihydroxylated and the 1,2-diol was oxidatively cleaved with sodium metaperiodate. Formation of the corresponding dibromoolen was then followed by palladium-catalyzed trans-selective ethynylation-methylation tandem cross coupling, delivering the 1,3-enyne 92 as a unique isomer in 61% yield aer removal of the acetylenic trimethylsilyl

Scheme 15 Two generations of syntheses of mycolactone A C10 –C70 fragment (eq. 1 and eq. 2) and synthesis of mycolactone B C10 –C70 fragment (eq. 3). Reagents and conditions: (1) n-BuLi, THF; (2) TMSCl; (3) HCl; (4) (COCl)2, DMSO; (5) CBr4, PPh3, Zn, 90%; (6) Me2Zn, Pd(dpephos)Cl2 (5 mol%), DMF/THF, 20 C, 70%; (7) 87, Pd(dpephos)Cl2 (5 mol%), Et2O/THF/DMF, 20 to 45 C, 82%; (8) TBAF, THF, 20 C, 81%; (9) BBr3, CH2Cl2, 78 to 20 C; (10) pinacol, CH2Cl2, 78 to 20 C; (11) 88, dichloro[1,3-bis(2,6-di-3-pentylphenyl)imidazol-2-ylidene](3-chloropyridyl) palladium(II) (PEPPSI) (1 mol%), THF, 20 C; (12) I2, NaOH, THF/H2O, 77%; (13) Et2Zn, THF, 0 C; (14) 89, Pd(tBu3P)2 (0.5 mol%), THF, 20 C, 94%; (15) AlMe3, Cp2ZrCl2 (30 mol%), CH2Cl2, 78 to 0 C; (16) I2; (17) TBSCl, imidazole, DMF, 20 C, 65%; (18) i-Bu2AlH, CH2Cl2, 78 to 0 C; (19) i-Bu2AlH, Cp2ZrCl2 (120 mol%), CH2Cl2, 0 C; (20) I2, 78 C, 74% (3 steps); (21) Et2Zn, THF, 0 C; (22) 89, Pd(dpephos)Cl2 (5 mol%), THF, 0 to 20 C, 58%; (23) Me3Al, Cp2ZrCl2 (50 mol%), CH2Cl2, 78 to 0 C; (24) I2, 78 C, 70% (2 steps); (25) Et2Zn, THF, 0 C; (26) 89, Cl2Pd(dpephos) (5 mol%), THF, 0 to 20 C, 89%; (27) Me3Al, Cp2ZrCl2 (20 mol%), CH2Cl2, 78 to 0 C ; (28) I2, 78 C, 69%; (29) TBSCl, imidazole, DMF, 20 C, 63%.

Stereoselective syntheses of the two C10 –C70 fragments (geometrical isomers at the C40 –C50 p bond) are shown in Scheme 15 (eqs. 1 to 3). Two stereoselective syntheses159 of mycolactone A C10 –C70 fragment have been developed since the rst partial synthesis reported by Negishi in 2006 (Scheme 15, eq. 1 and eq. 2). The rst strategy (eq. 1)159a relied on the palladium-catalyzed trans-selective methylation-alkenylation tandem cross-coupling of 1,1-dibromo-1,3-enyne 82a, delivering


Scheme 16 Synthesis of mycolactone A/B C80 –C160 fragment. Reagents and conditions: (1) TBSCl, imidazole, DMF, 20 C, 97%; (2) i-Bu2AlH, CH2Cl2, 78 C, 85%; (3) 94, BF3$Et2O, Et2O, 90 to 0 C, 91%; (4) TBSOTf, 2,6-lutidine, CH2Cl2, 0 C, 100%; (5) OsO4 (1 mol%), NMO, THF/H2O, 20 C; (6) NaIO4, THF/H2O, 20 C, 98%; (7) PPh3, CBr4, 2,6-lutidine, CH2Cl2, 0 C, 96%; (8) 95, PdCl2(dpephos) (5 mol %), THF, 0 C; (9) Me2Zn, Pd(tBu3P)2 (2 mol%), THF, 20 C; (10) K2CO3, MeOH, 61%; (11) HCl (3M in H2O), MeOH, 55 C; (12) TBSOTf, 2,6-lutidine, CH2Cl2, 20 C, 64%; (13) HZrCp2Cl, THF, 20 C; (14) I2, 78 C, 85%.

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Scheme 17 Completion of Negishi's total synthesis of mycolactone A/B. Reagents and conditions: (1) tBuLi, Et2O, 78 C; (2) ZnBr2, Et2O/THF, 78 to 20 C (3) 93, 0 0 PdCl2(dpephos) (5 mol%), Et2O/THF/DMF; (4) TBAF, THF, 0 C; (5) Dess–Martin periodinane, CH2Cl2, NaHCO3; (6) NaClO2, NaH2PO4, tBuOH/H2O, 49% for 44-(Z-D4 -5 ), 40 -50 40 -50 40 -50 40 -50 40 -50 ); (7) 44-(Z-D ) or 44-(E-D ), Cl3C6H2COCl, DMAP, i-Pr2NEt, benzene, 20 C, 67% from 44-(Z-D ), 73% from 44-(E-D ); (8) TBAF, THF, 20 C; 44% for 44-(E-D 0 0 0 0 (9) AcOH/H2O/THF, 20 C, 41% from 44-(Z-D4 -5 ), 45% from 44-(E-D4 -5 ).

group. Protecting group swap and hydrozirconation/iodolysis nally furnished the dienyliodide 93 as a single enantiomer. Coupling of the two stereochemically pure C10 –C70 -fragments (40 Z)–86 and (40 E)–86 with the C80 –C160 -fragment 93 under Negishi conditions delivered a single isomer of the cor0 0 0 0 responding (Z-D4 -5 )- and (E-D4 -5 )-pentaene (Scheme 17, eq. 1) whose structure was proved by nOe analysis. Aer selective deprotection of the primary silylethers, a two-step sequence nally gave the desired pentaenic carboxylic acids 44 ready for the esterication reaction with the C1–C20 fragment 45 of mycolactone A/B. The nal esterications were performed following Kishi's lead under Yamaguchi conditions, leading to fully protected mycolactone A and mycolactone B as single isomers according to 1H and 13C NMR analysis (eq. 2). A twostep sequence for deprotection was nally applied, the silylethers being removed rst (inducing an isomerization of the C40 –C50 p bond) followed by treatment with an aqueous acetic acid solution. Mycolactones A/B from both routes were then isolated as a thermodynamically equilibrated mixture of 1 : 0.75–0.82, thus completing this strategically distinct approach to these seducing toxins.

8.4

Total synthesis of mycolactone A/B by Altmann (2011)

In 2011, Altmann160 disclosed the third total synthesis of mycolactone A/B with the specic aim of providing pure synthetic toxins for biological studies, including the production of antibodies against mycolactone and the study of structure– activity relationships, as in the synthesis of simplied mycolactone analogues by Blanchard that appeared shortly aerwards.138 Altmann's synthetic blueprint called for the bond

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disconnections proposed by Kishi excepted for the elaboration of the macrolactone that relied on a (E)-selective ring-closing metathesis reaction for the creation of the (E)-trisubstituted C8–C9 alkene (Chart 1, Box 3, Section 8.1). For the different fragments of mycolactone A/B, Altmann combined synthetic approaches already reported for the elaboration of the C10 –C160 and C14–C20 portions of the toxins that are detailed in the Electronic Supplementary Information section or that have been presented earlier, respectively. Worthy of note is the strategy for the construction of the alkenyl motif of the undecenolide that relied on a RCM, a strategy pioneered by Burkart161 and also adopted by Blanchard in a simplied version (see also section 9.2).138 It is interesting to note that such a strategy for the construction of trisubstituted alkenes embedded in twelve-membered lactone is quite difficult and therefore uncommon in the literature.162 Only a handful of examples could be found that all rely on a preorganization of the RCM-precursor via a 1,2-disubstituted aromatic ring, highlighting once again the subtleties of metathetic reactions.163 The required metathesis precursor has been prepared via esterication of the carboxylic acid C1–C8 fragment 99 (Scheme 18, eq. 1) with the homoallylic alcohol 102 (eq. 2) corresponding to the C9–C13 fragment. The former fragment was prepared from 1,5-pentanediol 67 that was mono-protected as a p-methoxybenzyl ether followed by a Swern oxidation and an Oppolzer aldolisation reaction with sultam 100 (Scheme 18, eq. 1). Diol 96 was obtained in good yield with complete diastereoselectivity, aer reduction of the acylsultam with lithium aluminum hydride. The stage was then set for an elegant formation of oxetane 97 via an intramolecular SN2 process, using tosylation of 96 followed by treatment with sodium


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Scheme 18 Altmann syntheses of the C1–C8 (eq. 1) and C9–C13 (eq. 2) fragments. Reagents and conditions: (1) NaH, PMBCl, THF, reflux; (2) (COCl)2, DMSO, Et3N, CH2Cl2, 78 to 20 C; (3) 100, 78 C, CH2Cl2, 78 C; (4) LiAlH4, THF/Et2O, 0 to 20 C, 62%; (5) TsCl, DMAP, Et3N, CH2Cl2, 0 to 20 C; (6) NaH, THF, 20 to 40 C, 94%; (7) isopropenyllithium, BF3$OEt2, Et2O, 78 C; (8) TESOTf, 2,6-lutidine, CH2Cl2, 78 to 20 C, 88%; (9) DDQ, pH 7.2 buffer, CH2Cl2, 20 C; (10) Dess– Martin periodinane, CH2Cl2, 0 C; (11) NaClO2, 2-methyl-2-butene, NaH2PO4, t BuOH, H2O, 20 C, 84%; (12) 4-methoxybenzyl-2,2,2-trichloroacetimidate, TfOH, Et2O, 20 C; (13) DIBAL-H, CH2Cl2, 78 C, 58%; (14) allyltributylstannane, SnCl4, CH2Cl2, 90 C, 81%; (15) DDQ, CH2Cl2, H2O; (16) LiAlH4, Et2O, 0 to 20 C; (17) TsCl, Et3N, DMAP, CH2Cl2, 35 C, 65%.

hydride. This oxetane could be ring-opened with an excess of isopropenyllithium, thus leading to 98. Protecting group manipulation followed by oxidation of the C1-primary alcohol into the corresponding carboxylic acid nally delivered the required C1–C8 fragment 99 of mycolactone A/B on a multigram scale. On the other hand, fragment C9–C13 102 was prepared from methyl (S)-(+)-3-hydroxy-2-methylpropionate 101 in six steps (eq. 2) thanks to diastereoselective allylation of the intermediate aldehyde with allyltributyltin, followed by classical functional group manipulations. Finally, union of the two fragments under H¨ oe–Steglich conditions offered the RCM precursor in 82% yield (Scheme 19). The outcome of the crucial RCM reaction proved to be highly capricious under otherwise similar reaction conditions with the reaction time for complete conversion of the RCM precursor being comprised between 2.5 and 24 h. Similar observations were made during the synthesis of a library of analogues with very closely related substrates (see Section 9). In the best cases, the RCM reaction in the presence of 12 mol% of Grubbs-2 catalyst in reuxing dichloromethane at 3 millimolar led to 80% isolated yield of the desired (E)-trisubstituted undecenolide. As noted by Altmann, production of the corresponding (Z)-isomer could not be disproved but was not observed in the isolated


NPR

Scheme 19 Completion of Altmann’s total synthesis of mycolactone A/B. Reagents and conditions: (1) 102, DCC, DMAP, CH2Cl2, 20 C, 82%; (2) [Ru]-2 (12 mol%), CH2Cl2, (3 mM), reflux, 80%; (3) NaI, acetone, 60 C, 95%; (4) tBuLi, 9-MeO-BBN, Et2O, THF, 78 to 20 C; (5) 104, PdCl2(dppf) (10 mol%), AsPh3 (30 mol%), Cs2CO3 (3M in H2O), DMF, 20 C, 80%; (6) AcOH/H2O/THF, 20 C, 90%; (7) 44, Cl3C6H2COCl, i-Pr2NEt, DMAP, THF, 20 C, 89%; (8) HF$pyridine, THF/pyridine, 20 C, 84%; (9) TBAF, THF, 20 C, 85%.

fractions. Finally, the C13-tosyloxy group of macrolactone was converted to the primary iodide 103 with sodium iodide in reuxing acetone. The last steps of Altmann's total synthesis of mycolactone A/B relied on a modied Suzuki cross-coupling of the C1–C13 and C14–C20 fragments in 80% yield followed by a high-yielding selective deprotection of the C5-silylether under acidic conditions. Esterication with the C10 –C160 pentaenoic acid fragment followed by stepwise deprotection of the cyclic C17,C19-bis-silylether and then of the C120 ,C130 ,C150 -tri silylethers offered fully deprotected mycolactone A/B in 71% yield 0 0 0 0 as a E-D4 ,5 :Z-D4 ,5 ¼ 1 : 1 mixture plus an additional 10% of other geometrical isomers. Reverse-phase HPLC nally delivered synthetically pure mycolactone A/B as a mixture of geometrical isomers, in 45% yield and 95% purity. 8.5

Photochemical behavior of mycolactone A/B (2012)

In 2012, Kishi reported an insightful study on the photochemistry of mycolactone A/B that uncovered elegant and selective transformations of the unsaturated fatty acid side chain.79 Intrigued by a complete loss of toxicity of mycolactone A/B

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NPR against keratinocytes upon exposure to light for half an hour that was observed at the Karolinska Hospital of Stockholm, an in-depth study was conducted using irradiation of synthetic mycolactone A/B and of model substrates by UV lamp equipped with a 365 nm lter and natural visible light. Irradiation of synthetic mycolactone at 365 nm in a 30 C acetone solution led to a mixture of four closely related compounds possessing the C20 –C30 and C100 –C110 olens intact (Scheme 20, eq. 1). Although the hypothesis of a [4p + 2p] cycloaddition of the C40 – C90 trienic motif leading to a bicyclo[3,1,0]hexene scaffold seemed plausible, the different isomers were difficult to separate. Thus, the photochemical behavior of tetraenoates 106a–d was studied as a model system.

Review Upon irradiation of tetraenoate 106a at 365 nm for 2.5 h, a mixture of four geometrical isomers was formed in a (40 E,60 E,80 E,100 E)-106a/(40 Z,60 E,80 E,100 E)-106b/(40 E,60 Z,80 E,100 E)106c/(40 Z,60 Z,80 E,100 E)-106d ¼ 2 : 7 : 1 : 1 ratio, the structure of which being determined by NMR experiments (Scheme 20, eq. 2). This photochemical steady state evolved slowly with time through a [4p + 2p] cycloaddition of the C40 –C90 triene and aer two days at 30 C, a mixture of four bicyclic products was obtained. Aer separation by chromatography on silica gel chromatography, uoride-mediated deprotection of the silyl ethers and HPLC purication on a chiral phase, 107a–d were obtained as homogeneous compounds. These diastereomers could be classied in two sub-groups, namely A and B,

Scheme 20 The photochemical transformation of mycolactone A/B (eq. 1) and of model tetraenoates (eq. 2). Reagents and conditions: (1) hn (365 nm), acetone, 30 C; (2) hn (365 nm), acetone, 30 C, 2.5 h; (3) hn (365 nm), acetone, 30 C, 2 days; (4) silica gel chromatography; (5) TBAF, THF, 20 C; (6) HPLC separation on chiral phase.

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NPR

Scheme 21 Conversion of model photoproducts to photomycolactones. Reagents and conditions: (1) TBSCl, AgNO3, pyridine, DMF, 94%; (2) DIBAL-H, CH2Cl2, 78 C, 90%; (3) Dess–Martin periodinane, NaHCO3, CH2Cl2, 84%; (4) methyl (triphenylphosphoranylidene)acetate, toluene, 90 C, 87%; (5) NaOH, THF/MeOH/H2O, 20 C, 90%; (6) 55, Cl3C6H2COCl, i-Pr2NEt, DMAP, toluene, 20 C, 91%; (7) TBAF, imidazole$HCl, THF, 20 C, 6 days, 96%.

differing only by the relative conguration of the C40 and C70 ,C80 -stereocenters. Their relative and absolute stereochemistries were determined via a combination of chemical correlation, NMR experiments and a X-ray structure of the trip-bromobenzoate of 107a-A1. In addition, it was found that the photocycloaddition reaction occurred at the singlet state since triplet quenchers such as azulene, ferrocene or rubrene did not affect the transformation. All these informations point towards a concerted and photochemically allowed [4ps + 2pa] cycloaddition. Functional group transformations of 107a led to the C10 – C160 fragment 108a that was further advanced to the synthetic photo-mycolactone 109a-A1 (Scheme 21). Similar synthetic transformations of 107b–d delivered the three complementary synthetic photomycolactones 109b–d. Finally, the identity of the photoadducts obtained from mycolactone A/B (Scheme 21) was established via comparison with the unique 1H NMR data and HPLC retention time of the four synthetic photomycolactones 109a–d. As seen in this section, the discovery of mycolactone A/B in 1999 has spurred a lot of interest from the synthetic community and elegant total syntheses started to appear very rapidly at the beginning of the 2000's with the seminal work of Kishi. Besides the total syntheses of the mycolactones, the modular preparations of modied, truncated or uorescent analogues have been recently reported to explore the fascinating biology of mycolactones that is still poorly understood from a molecular perspective. These reports are discussed in the next section.

9 Synthesis of mycolactone analogues and structure–activity relationship studies Conceptually related to the total synthesis are the chemical biology studies of Altmann,111,160 Kishi152 and Blanchard138 independently reported in 2011 that aim at deciphering the complex biology of these macrolides using chemical strategies. These rst structure–activity relationships (SAR) studies of This journal is ª The Royal Society of Chemistry 2013

mycolactone A/B served also as a guide for the design of uorescent mycolactone probes, that were essential in the conrmation of the rst proteic target of mycolactone, reported by Demangel139 in 2013 (see Section 7.4), thus highlighting the power of molecular editing in the investigation of the biology of complex natural products.164 This section will discuss the synthetic routes rst towards mycolactone analogues (Sections 9.1 and 9.2), then towards their uorescent derivatives (Section 9.3). A discussion of the SAR studies will close this report (Section 9.4). 9.1

Synthesis of mycolactone A/B analogues

From an historical perspective, in their seminal 1999 disclosure of mycolactone A/B, the Small group reported several chemical derivations of these natural toxins, such as the peracetylation of the hydroxyl groups or the hydrogenation of the double bonds.74 All these structural modications had a dramatic impact on the biology of the toxins, as discussed in Section 9.4. In addition, and from a synthetic perspective, the early efforts of Kishi should be recalled since numerous possible stereoclusters of mycolactones were synthesized in 2001 for the elucidation of the stereostructure of mycolactone A/B.152 Altmann reported the rst mycolactone analogues that were specically designed and biologically evaluated late 2011 (Scheme 22, eq. 1).160 The analogue 111 was prepared by reduction of tosyloxy-macrolactone 110 with sodium borohydride in dimethylsulfoxide at 100 C followed by deprotection under acidic conditions. Esterication of 111 with sorbic acid led to the truncated analogue 112, lacking the C14–C20 fragment of mycolactone A/B. In early 2013,111 six new analogues 113–117 were reported by the same group following a similar synthetic strategy (Scheme 22, eqs. 2 and 3). Finally, the two analogues of mycolactone A/B 118a and 118b, modied in the fatty acid side chain portion, were reported in a review article by Kishi in 2011 (eq. 3).152 The biology of all these derivatives will be presented in Section 9.4.

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the fatty acid side chain. For the elaboration of the macrolactonic portion of these analogues, the key disconnections reported by Kishi152 for the C13–C14 s bond and the RCM approach pioneered by Burkart161 for the elaboration of the disubstituted C8–C9 p bond were adopted (see Chart 1, Box 3, Section 8.1). Recognition of the identical stereochemistry at C6 and C12 led to a simplication of the synthetic scheme (Scheme 23). Following the lead of F¨ urstner, methyl (S)-(+)-3-hydroxy-2methylpropionate 101 was converted into the b-tosyloxy aldehyde 119, in which the tosyl group acts both as a protecting group and a leaving group in a subsequent step (eq. 1). Brown's allylboration165 using both enantiomers of commercially available allyldiisopinocampheylboranes afforded homoallylic alcohols 102-syn and 102-anti as single diastereomers on a multigram scale. The syn-diastereomer was homologated to

Scheme 22 Mycolactone analogues by Altmann. Reagents and conditions: (1) NaBH4, DMSO, 100 C, 76%; (2) AcOH/THF/H2O, 20 C, 98%; (3) sorbic acid, EDCI, DMAP, CH2Cl2, 20 C, 99%.

9.2

Synthesis of C8-desmethyl mycolactone A/B analogues

Almost concomitantly with Altmann's and Kishi's reports, Blanchard disclosed a modular synthesis of nine mycolactone analogues (including a uorescent one) whose CPE were systematically investigated on L929 broblasts.138 From a synthetic perspective, three areas of structural modications were specically targeted: simplication of the C14–C20 fragment, reduction of the substitution of the C8–C9 p bond and variations in the C120 ,C130 ,C150 -stereocluster, including the synthesis of all possible diastereomers as well as their monoand dideoxy counterparts. The synthetic blueprint relied on the classical esterication reaction of the appropriately protected C1–C20 fragment with

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Scheme 23 Synthesis of the C1–C20 fragment. Reagents and conditions: (1) TsCl, Et3N, DMAP, CH2Cl2; (2) DIBAL-H, toluene, 87% (2 steps); (3) TEMPO (10 mol %), PhI(OAc)2, CH2Cl2; (4) ()- or (+)-Ipc2Ballyl, Et2O, 78 C, 70% with d.r > 97 : 3 for 102-syn, 76% with d.r. > 97 : 3 for 102-anti; (5) NaBO3$4H2O; (6) TBSCl, imididazole, CH2Cl2, 95%; (7) acrylic acid, [Ru]-2 (3 mol%), CH2Cl2, 90 C (MW); (8) H2, Pd(OH)2, EtOAc, 79% (2 steps); (9) NaI, acetone, 90%; (10) vinylmagnesium bromide, FeCl3 (20 mol%), TMEDA, THF, 0 C, 51%; (11) 102-anti, DCC, DMAP, CH2Cl2, 82%; (12) [Ru]-2 (10 mol%), CH2Cl2, 90 C (MW), 83%; (13) NaI, acetone, 92%; (14) Rieke zinc, THF, 20 C; (15) 49, Pd(PPh3)4 (13 mol%), LiCl, NMP/DMF/ C6H6, 55 C, 63%; (16) HF$pyridine, THF–pyridine, 72% (brsm).


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Review carboxylic acid 120 in a three-step sequence based on a ruthenium-catalyzed cross-metathesis with acrylic acid followed by hydrogenation over palladium hydroxyde and Finkelstein reaction of the C7-tosyloxy group with sodium iodide (eq. 2). The primary iodide 120 was then submitted to an iron-catalyzed vinylation reaction using the conditions reported by Cossy,166 which delivered 121 in moderate yield. It is worth noting that this iron-catalyzed strategy allowed C1 to remain at the acid oxidation state, which saved the classical three-step deprotection/oxidation sequence. Esterication of the C1–C8 fragment 121 with the diastereomer 102-anti led to the RCM precursor that could be efficiently cyclized to a single isomer of the desired twelve-membered macrolactone. Aer a last Finkelstein reaction of the C13-tosyloxy group, the iodo-macrolactone 122 was obtained in 76% yield for two steps. Conversion

Scheme 24 Synthesis of C8-desmethyl mycolactone A/B. Reagents and conditions: (1) NaH, CHI3, Et2O; (2) KOH, EtOH, H2O, reflux, 43%; (3) LiAlH4, THF, 75%; (4) MnO2, CH2Cl2; (5) (carbethoxyethylidene)triphenylphosphorane, CH2Cl2, 0 to 20 C, 76%; (6) DIBAL-H, CH2Cl2, 0 C, 95%; (7) MnO2, CH2Cl2; (8) (carbethoxymethylene)triphenylphosphorane, CH2Cl2, 100%; (9) AD-Mix a, K2OsO4$2H2O (0.6 mol%), MeSO2NH2, tBuOH–H2O, 0 C, 70%, 86% ee; (10) (Cl3CO)2CO, pyridine, CH2Cl2, 79%; (11) Pd2(dba)3$CHCl3 (0.5 mol%), Et3N, HCO2H, THF, 20 C, 63%; (12) TBSCl, imidazole, DMF, 93%; (13) AD-Mix a, K2OsO4$2H2O (2 mol%), MeSO2NH2, tBuOH–H2O, 0 C, 70%; (14) TBSCl, imidazole, DMF, 83%; (15) DIBALH, CH2Cl2, 78 C, 97%; (16) MnO2, CH2Cl2, 94%; (17) CrCl2, n-Bu3SnCHBr2, LiI, THF–DMF, 20 C; (18) 127, CuTC, Ph2P(O)OBu4N, NMP, 20 C, 48% (2 steps); (19) LiOH, THF–H2O, 92%; (20) hn (12 mW cm2), acetone, 100%; (21) 124, i-PrNEt2, Cl3C6H2COCl, DMAP, C6H6, 82%; (22) TBAF, THF, 34%.


NPR to the full C1–C20 fragment then followed Kishi's strategy with only minor variations. Selective deprotection of the C5-silyloxy group could be performed using the hydrogen uoride pyridine complex in 72% based on recovered starting material. The fatty acid side chain of mycolactone is the only structural area of variation among all the members of this family of toxins, thus indicating that the major differences in cytopathicity could be explained by these structural differences. Logically, a modular approach that would expedite the synthesis of analogues was highly desirable. A strategy based on a copper(I)mediated Stille coupling between the (conserved) C10 –C70 fragment and a diversity of C80 –C160 fragments was therefore proposed. Fragment C10 –C70 127 was prepared in eight steps from diethyl methylmalonate 125 (Scheme 24, eq. 1). The sodium enolate of the latter was alkylated with iodoform and then saponied with potassium hydroxide in reuxing aqueous ethanol. A spontaneous decarboxylation delivered (E)-3-iodo-2methacrylic acid 126 in 47% yield for two steps that was next reduced with lithium aluminum hydride in 75% yield. This three-step sequence from 125 was found to be more practical than the single-step Negishi methylalumination (with neat trimethylaluminum)/iodolysis of propargylic acid on large scale. Further oxidation, olenation and reduction afforded (2E,4E)-5iodo-2,4-dimethylpenta-2,4-dien-1-ol in good overall yield. A nal oxidation of the primary alcohol with manganese dioxide followed by Wittig olenation gave the C10 –C70 -fragment 127. Although the overall sequence is quite linear, its main advantages are an easy scale-up (routinely on a decagram scale) and the use of non-hazardous and inexpensive reagents. As mentioned above, the synthesis of the second fragment of the fatty acid side chain heavily relied on catalytic asymmetric transformations (Scheme 24, eq. 2). Starting from trienoate 128, a catalytic asymmetric dihydroxylation proceeded with a complete chemoselectivity and a good enantioselectivity, delivering the corresponding diol 129a in 86% ee. Formation of the carbonate delivered 129b in 79% yield and allylic reduction of the latter using palladium(0) and formate followed by silyl ether protection gave the desired ester 130 in a good overall yield. This two step-sequence from diol has been proposed by O'Doherty167 and was especially interesting in the context of the preparation of analogues. Further asymmetric dihydroxylation of 130 occurred with complete chemo- and diastereoselectivities, leading aer protection to compound 131 that possessed the C120 ,C130 ,C150 -stereocenters of natural mycolactone A/B. Two trivial steps converted the ester moiety to the corresponding aldehyde that was further processed to the (E,E)dienylstannane 132 using a chromium-mediated one carbon homologation reported by Hodgson.168 For the strategic cross-coupling of the C10 -C70 - and C80 –C160 fragments, a copper(I)-mediated reaction was chosen based on efficiency and also practical considerations.169 Using copper(I) thiophenecarboxylate and tetrabutylammonium diphenylphosphinate as a tin salt sequestrant, a smooth coupling occurred leading to the desired pentaene with the correct 0 0 oxidation state at C10 in 48% yield for two steps, as a Z-D4 ,5 :E0 0 D4 ,5 ¼ 20 : 80 mixture. To the best of our knowledge, the use of

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Chart 2

Synthesis of C8-desmethyl mycolactone A/B analogues.

a CuTC-mediated cross-coupling for the elaboration of a pentaene motif is unprecedented. Saponication of the ester followed by photochemical equilibration using a standard green house bulb (12 mW cm2) delivered acid 44 as a 0 0 0 0 Z-D4 ,5 :E-D4 ,5 ¼ 60 : 40 mixture. Esterication of the latter with the C1–C20 fragment 124 followed by uoride-mediated deprotection of the ve silyl ethers nally delivered C8-desmethyl mycolactone A/B 133. This exible synthetic strategy has been applied to the preparation of four C8-desmethyl mycolactone analogues 133a– d and three truncated analogues 134, 135 and 136 (Chart 2). Pentaenic acid 144 was also esteried with isopropanol leading to the isopropyl ester 137. Before discussion of the biological evaluation of these mycolactone analogues and to close this synthetic section, the preparation of the uorescent mycolactones analogues will be presented in the next paragraphs. 9.3

Synthesis of uorescent mycolactone analogues

In 2003, Small reported on the synthesis and use in cell biology of the rst BODIPY-derived mycolactone 138b.137 The latter was obtained in two steps from cultured natural mycolactone A/B by periodic acid cleavage of the C120 ,C130 -diol motif followed by conjugation of the intermediate C120 -aldehyde 138a with Invitrogen's Bodipy FL D-2371 (Scheme 25, eq. 1). Purication on a C18 solid-phase extraction cartridge nally delivered bodipymycolactone 138b that produced the same phenotype as natural mycolactone A/B in cultured cells, although the cytopathic activity was slightly reduced (minimum effective dose of 130 nM

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Scheme 25 Synthesis of fluorescent mycolactone A/B analogues. Reagents and conditions: (1) HIO4, HCl, EtOH, 37 C; (2) Bodipy FL hydrazide (D-2371), CHCl3, MeOH; (3) NaN3, DMF, 75 C, 79%; (4) TBAF, THF, 74%; (5) 44, i-PrNEt2, Cl3C6H2COCl, DMAP, C6H6, 85%; (6) TBAF, THF, 20 C, 93%; (7) 4,4-difluoro-8(hept-6-yne)-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-S-indacene, Cu(OAc)2$H2O (15 mol%), NaAsc (15 mol%), tBuOH-H2O, 60 C, 35%.

for 138b versus 20 nM for mycolactone A/B). Confocal microscopy on cultured broblasts incubated with 138b demonstrated that a passive diffusion of the uorescent toxin occurred, the latter being excluded from the nucleus and localized only in the cytosol without binding to internal membranes as shown by photobleaching experiments. Non-signicative co-localizations with the mitochondria and actin were also noted, which appeared surprising in the latter case since mycolactone A/B is known to induce cytoskeletal rearrangement rapidly. Considering these results, Small postulated that bodipy-mycolactone 138b might bind slowly and irreversibly to cytoplasmic proteins. This 2003 report clearly demonstrated that the design of


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Review relevant uorescent probes is an important tool for exploring the biology of complex natural products. To pursue Small's lead and based on the rst systematic structure–activity relationships studies that demonstrated the importance of the C120 ,C130 ,C150 -stereocluster, a synthetic approach in which a uorescent tag would be easily linked to the northern fragment of mycolactone analogues through a click chemistry strategy was developed by Blanchard (Scheme 25, eq. 2).138 To this end, macrolactone 139 was converted to the C13-primary azide followed by uoride-mediated deprotection of the C5-silyloxyether. The resulting alcohol 140 was esteried with the natural fatty acid side chain and the three silylethers were deprotected, thus leading to the [3+2] precursor 141. Upon copper(I)-catalyzed cycloaddition with a known u-alcynylBODIPY derivative, the uorescent analogue 142 was obtained, possessing a maximum absorbance at 505 nm.

9.4

Structure–activity relationship studies

As discussed in Section 8 and in the ESI,† several research groups have developed efficient partial and total syntheses of mycolactones (A/B, C, E, F, dia-F, S1, S2), a family of toxins that differ only by the nature of the C10 –Cn0 fatty acid side chain (Fig. 9, Section 6.4). Since the seminal discovery of mycolactone

Fig. 12

NPR A/B by Small in 1999,74 important informations regarding the structure–activity relationships of this beautiful family of toxins have been reported in the literature and this last sub-section aims at proposing an overview and a discussion of these scattered informations. 9.4.1 Naturally occurring mycolactones. We shall open this sub-section by recalling the known data of cytotoxic and immunosuppressive activities of the nine different naturally occurring mycolactones that infect humans (mycolactones A/B, C, D, S1 and S2), frogs (E and E-minor metabolite), salt-water sh (F) and fresh-water sh (dia-F) (Fig. 12) (see also Section 7). Among the different toxins infecting humans, mycolactone A/B are the most active, with a reported concentration necessary for cytopathicity as low as 25 pg mL1, in the initial 1999 report by Small and collaborators.74 Recent detailed investigations using ow cytometry have estimated the concentration of mycolactone for which half of the cells were killed (LC50) and for which the metabolic activity of the cells was reduced by half (IC50). The LC50 was estimated at 12 nM, the IC50 at 5 nM and the LC50/IC50 ratio at 2.4.111 In 2003, natural mycolactone C was shown to be 104 less active than the parent A/B,108 which was further conrmed with synthetic mycolactone C.110 However, different results were reported in 2013 using ow cytometric analyses of murine broblast L929 cells since a LC50 of 186 nM

An overview of known biological activities of the mycolactones.


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NPR was found, which corresponds to a difference of only 15.5 compared to mycolactone A/B.111 With a cytopathic activity of 100 ng mL1 (15 nM), the frog toxin mycolactone E was found to be 100 times less cytopathic than mycolactone A/B on L929 broblasts,115 while the GI50 of the mycolactone E-minor metabolite was measured at 15 nM, a value similar to the one of mycolactone E.117 Finally, the sh pathogens mycolactone F and dia-F present a strikingly different cytotoxicity since the F congener was 103 more potent than the dia-F one,122 thus stressing the importance of the absolute congurations of the C110 and C130 stereocenters. With a LC50 of 29 nM, mycolactone F was still 2.4 less potent than mycolactone A/B.107 Mycolactone G (although not of natural origin) has no detectable cytotoxicity on Jurkat T-cells even at high concentration (up to 1 mg mL1 for 24 h).121 A comparison of the effect of natural mycolactones A/B, C, E, F and of the genetically engineered mycolactone G on the production of IL-2 was reported in 2007.121 This study shown that mycolactone A/B was the most active inhibitor of IL-2 production while the G congener was the least active. A last point that ought to be recalled on naturally occurring mycolactone A/B is the absence of any activity against several gram-positive and gram-negative microbial species as well as against yeast and amoeba (see Section 7 for details).111 9.4.2 Chemical modications of naturally occurring mycolactone A/B. Historically, the rst structural modications of mycolactone A/B were briey mentioned by Small and collaborators in 1999, using the toxin isolated from Mycobacterium ulcerans cultures.74 As mentioned in Section 9.1, peracetylation of mycolactone A/B as well as hydrogenation of all the double bonds were reported to induce the loss of the cytopathic effect, although no specic data were reported. In 2003, the rst hints on the importance of the fatty acid side chain for cytotoxicity began to be reported.108 A truncated mycolactone A/B, obtained by basic hydrolysis of the natural toxin, was evaluated for CPA and it was found that the C1–C20 fragment was 104 less potent than the natural toxin, although a similar phenotype was observed on L929 broblasts. In a separate publication, Small reported two unnatural mycolactones obtained by chemical modications of the southern fragment: a C120 -aldehydic mycolactone and a uorescent mycolactone probe that did not contain the C120 –C160 -motif (see Section 9.3).137 The CPA of these compounds was estimated to be 190 and 130 nM, respectively, which corresponds to values 10 and 6.5 times inferior to the one of mycolactone A/B. Although Small suggested that the C130 –C160 subunit was “an inactive hydrophilic portion” of the toxin and could therefore be chemically manipulated, all these data point towards the crucial importance of the C10 –C160 fatty acid side chain for the cytotoxic activity, a quite logical assumption since the different mycolactones present a broad spectrum of toxicity on humans, sh or frogs and differ only in the nature of this C10 –Cn0 fragment (see Section 6.4). It is important to note that the biology of the naturally occurring mycolactones could be difficult to investigate since the same microorganism can produce several mycolactones.

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Review Such an example can be found with the Australian strains that produce a mixture of mycolactones A/B and C, the latter being the major component. In addition, problems of chemical purity of the toxins isolated from natural sources are recurrent. To overcome these problems of structural heterogeneity and purity, chemical synthesis is a very efficient tool that has been crucial in recent studies of structure–activity relationships (SAR). 9.4.3 SAR studies based on de novo chemical syntheses. Up to now, only two SAR studies based on de novo chemical syntheses of mycolactone analogues have been reported in the literature by Altmann (8 analogues),111,160 and Blanchard (9 analogues).138 As we will see, Kishi also disclosed the biological activity of an isolated C160 -modied analogue of mycolactone A/B.152 Altmann and Kishi reported in 2011 and 2013 a series of mycolactone A/B analogues that can be classied in three categories (Scheme 22, see also Section 9.1).111,160 In the rst one, the C14–C20 northern fragment of mycolactone is removed and the C5-hydroxy group is either free (111) or acylated with sorbic acid (112). The second class comprises modications at C20, such as the C20-hydroxy derivative (113), the C20-hydroxyacyl (114a) and the C20-butylcarbamate (114b). The last class of analogues includes the C1–C20 fragment of mycolactone A/B with the C5-hydroxy group esteried either with acetic acid (115), sorbic acid (116), with the C120 ,C130 ,C150 -trideoxy southern fragment of the natural toxin (117) or with C160 modied southern fragments (118a,b). For compounds 111–117, the apoptosis and necrosis of L929 broblasts was studied, as well as the metabolic activity of cells aer incubation with mycolactones, using ow cytometry and uorescence microscopy (Fig. 13).111,160 No cytopathic effect was observed for the two simplied analogues 111 and 112, belonging to the rst class. In a similar vein, a signicant drop in cytotoxicity was found for 115–117 with LC50 values superior to 5000 nM for 115, of 3426 nM for 116 and of 4550 nM for 117 (compared to 12 nM for mycolactone A/B). The cytotoxicity of Kishi's derivatives are available only for 118b. The value of 30 nM compares very favorably with that of natural mycolactone A/ B (in the 10 nM range), demonstrating that extension of the southern fragment is possible without incidence of the CPE.152 On the other hand, a signicant cytotoxicity was conserved for the analogues of the third class with a LC50 of 15 nM for 117, similar to the one of the natural toxin, indicating that a polar hydroxy group at the C20 position is very well tolerated. Slightly higher LC50 values were measured for 118a (45 nM) and 118b (50 nM). For two selected derivatives (113 and 114), the inhibition of the proliferation of L929 broblasts was investigated and it was found that only 114 displayed signicant inhibition aer 72 h. Finally, 114 also induced a transient effect on the actin cytoskeleton as well as a classical rounding up of the broblasts. In 2011, Blanchard and coworkers reported the cytopathic effects of nine C8-desmethylated analogues on L929 murine broblasts, by determination of the percentage of cell rounding.138 Four classes of analogues were evaluated: the rst class includes the C8-desmethyl C1–C20 fragment whose C5-hydroxy group is either free (134) or acylated with diastereomeric C10 –C160 fatty acid side chains (133a–d), 133a corresponding to This journal is ª The Royal Society of Chemistry 2013

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Fig. 13 An overview of known biological activities on L929 fibroblasts of the different mycolactone analogues. a Cytopathic activity as the minimum concentration for 90% of cell rounding b LC50 after 48 h of incubation c Percentage of cell rounding at 10 mM after 48 h of incubation.

NPR results are qualitatively in agreement with those obtained by Altmann, pointing towards the tolerance of the modications in the northern fragment and the crucial importance of the southern fragment. The next step in this SAR studies was to evaluate 133a–d, that differ only by the nature of the C120 –C150 stereocluster. At 10mM, 100% of cell rounding was observed for the structurally closest analogue of the natural mycolactone A/B, 133a. In addition, the biological importance of the C120 -hydroxyl was conrmed, with 49% of cell rounding at 10 mM for compound 133d. This important decrease in activity is logical since the same trend was observed between mycolactones A/B and C. The complete inversion of the C120 –C150 stereocluster as in 133c led to a dramatic loss of CPE, with only 10% of cell rounding at 10 mM. However, the epi-C150 analogue 133b showed 100% of cell rounding, suggesting that the C150 position is less important for CPA. Finally the lowest concentration inducing 90% of cell rounding, a standard evaluation for mycolactone CPE, was evaluated for 133a at 5 mM, to be compared with the 40 nM found for the natural mycolactone A/B in the same test. This C8-desmethylated analogue is therefore 125 less cytopathic than the natural mycolactone, thereby demonstrating the importance of the C8 methyl group. To close this section, the relevance of the uorescent derivative 142 will be discussed. The CPE of 142 was not different from the parent derivative 133a since 90% of cell rounding was observed at 10 mM aer 48 h. More importantly, no cell rounding was noted at 0.5 mM, the concentration of the uptake experiments. These cellular uptake studies conrmed a rapid passive diffusion through cell membranes and an exclusive cytosolic localization in agreement with Small's studies.137 Finally, this uorescent probe was one of the keys to the conrmation of the rst identied protein target of mycolactone A/B, the Wiskott–Aldrich Syndrom protein,139 as discussed in Section 7.4. Overall, within the last ten years, the accumulated knowledge in mycolactone chemical biology clearly points towards the importance of the C10 –C160 portion of the mycolactone that contains the crucial C120 –C150 -stereocluster. In addition, the developments of tailored-made mycolactone probes have been central to the search of the molecular target(s) of these potent toxins.

10 the fatty acid side chain of natural mycolactone A/B, the second class of analogues encompasses two C14–C20 truncated toxins having the C5-hydroxy group either free (135) or acylated with the natural fatty acid side chain (136), the third class includes a single derivative of the southern fragment (137) and eventually, the last class concerns the uorescent derivatives such as 142. The least potent derivatives were 135 and 137, since no cytopathicity could be detected at 10 mM aer 48h (and only less than 20% of cell rounding at 50 mM). Removal of the southern (134) of northern fragment (136) of C8-desmethyl mycolactone A/B resulted in a dramatic drop in CPA with less than 29 and 53% of cell rounding at 10 mM aer 48h, respectively. Theses


Conclusions

Although M. ulcerans infections in humans (Buruli ulcer disease) have been reported for more than 150 years, the mechanistic understanding of this unusual necrotic skin disease is quite recent. The discovery of the causative mycobacteria in 1948 and of the secreted pathogenic agent, mycolactone A/B in 1999, are clearly milestones that have changed the way we look at bioactive polyketides and their biological effects in humans. The intense research efforts from the biological and chemical communities have led to great advances in the past decade, which resulted in the understanding of the delicate interplay between biological effects and chemical structures. Multidisciplinary works focused on Buruli ulcers are

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essential in this respect as seen in this review. These investigations clearly pave the way for many more exciting developments in the mycolactone chemical biology.

11

Acknowledgements

The authors are truly indebted to Dr J. Hayman (Australia) for sharing unpublished reports and photographs, to Dr A. W. J. Logan for careful proofreading and to the referee for insightful comments. Financial supports from the Fondation Raoul Follereau, the CNRS, the University of Mulhouse and the University of Strasbourg are gratefully acknowledged.

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Experimental Survival of Mycobacterium ulcerans in Watery Soil, a Potential Source of Buruli Ulcer.

Seasonal Pattern of Mycobacterium ulcerans, the Causative Agent of Buruli Ulcer, in the Environment in Ghana.

Buruli ulcer disease.

Environmental transmission of Mycobacterium ulcerans drives dynamics of Buruli ulcer in endemic regions of Cameroon.

Locally Confined Clonal Complexes of Mycobacterium ulcerans in Two Buruli Ulcer Endemic Regions of Cameroon.

Burden of Mycobacterium ulcerans disease (Buruli ulcer) and the underreporting ratio in the territory of Songololo, Democratic Republic of Congo.

Spatial Distribution of Mycobacterium ulcerans in Buruli Ulcer Lesions: Implications for Laboratory Diagnosis.

Mycobacterium ulcerans persistence at a village water source of Buruli ulcer patients.

Mapping biopsy procedure on management of severe buruli ulcer due to Mycobacterium ulcerans, subspecies shinshuense.

Recombinant BCG Expressing Mycobacterium ulcerans Ag85A Imparts Enhanced Protection against Experimental Buruli ulcer.

Experimental infection of the pig with Mycobacterium ulcerans: a novel model for studying the pathogenesis of Buruli ulcer disease.

Mycobacterium ulcerans low infectious dose and mechanical transmission support insect bites and puncturing injuries in the spread of Buruli ulcer.

Pleiotropic molecular effects of the Mycobacterium ulcerans virulence factor mycolactone underlying the cell death and immunosuppression seen in Buruli ulcer.

Clinical considerations on Buruli ulcer employing two molecular tests for the detection of Mycobacterium ulcerans in 100 skin biopsies.

Local Cellular Immune Responses and Pathogenesis of Buruli Ulcer Lesions in the Experimental Mycobacterium Ulcerans Pig Infection Model.

Genetic diversity of PCR-positive, culture-negative and culture-positive Mycobacterium ulcerans isolated from Buruli ulcer patients in Ghana.

Analysis of Mycobacterium ulcerans-specific T-cell cytokines for diagnosis of Buruli ulcer disease and as potential indicator for disease progression.

Detection of Mycobacterium ulcerans subsp. shinshuense DNA from a water channel in familial Buruli ulcer cases in Japan.

Draft Genome Sequence of Mycobacterium ulcerans S4018 Isolated from a Patient with an Active Buruli Ulcer in Benin, Africa.

Evidences of the Low Implication of Mosquitoes in the Transmission of Mycobacterium ulcerans, the Causative Agent of Buruli Ulcer.

[Paradoxical reactions and responses during antibiotic treatment for Mycobacterium ulcerans infection (Buruli ulcer). Four cases from French Guiana].

Correction: Whole Genome Comparisons Suggest Random Distribution of Mycobacterium ulcerans Genotypes in a Buruli Ulcer Endemic Region of Ghana.

Probable Buruli Ulcer Disease in Honduras.

Source tracking Mycobacterium ulcerans infections in the Ashanti region, Ghana.