Exploring Mycolactone-The Unique Causative Toxin of Buruli Ulcer: Biosynthetic, Synthetic Pathways, Biomarker for Diagnosis, and Therapeutic Potential

Abstract

Mycolactone is a complex macrolide toxin produced by Mycobacterium ulcerans, the causative agent of Buruli ulcer. The aim of this paper is to review the chemistry, biosynthetic, and synthetic pathways of mycolactone A/B to help develop an understanding of the mode of action of these polyketides as well as their therapeutic potential. The synthetic work has largely been driven by the desire to afford researchers enough (≥100 mg) of the pure toxins for systematic biological studies toward understanding their very high biological activities. The review focuses on pioneering studies of Kishi which elaborate first-, second-, and third-generation approaches to the synthesis of mycolactones A/B. The three generations focused on the construction of the key intermediates required for the mycolactone synthesis. Synthesis of the first generation involves assignment of the relative and absolute stereochemistry of the mycolactones A and B. This was accomplished by employing a linear series of 17 chemical steps (1.3% overall yield) using the mycolactone core. The second generation significantly improved the first generation in three ways: (1) by optimizing the selection of protecting groups; (2) by removing needless protecting group adjustments; and (3) by enhancing the stereoselectivity and overall synthetic efficiency. Though the synthetic route to the mycolactone core was longer than the first generation, the overall yield was significantly higher (8.8%). The third-generation total synthesis was specifically aimed at an efficient, scalable, stereoselective, and shorter synthesis of mycolactone. The synthesis of the mycolactone core was achieved in 14 linear chemical steps with 19% overall yield. Furthermore, a modular synthetic approach where diverse analogues of mycolactone A/B were synthesized via a cascade of catalytic and/or asymmetric reactions as well as several Pd-catalyzed key steps coupled with hydroboration reactions were reviewed. In addition, the review discusses how mycolactone is employed in the diagnosis of Buruli ulcer with emphasis on detection methods of mass spectrometry, immunological assays, RNA aptamer techniques, and fluorescent-thin layer chromatography (f-TLC) methods as diagnostic tools. We examined studies of the structure-activity relationship (SAR) of various analogues of mycolactone. The paper highlights the multiple biological consequences associated with mycolactone such as skin ulceration, host immunomodulation, and analgesia. These effects are attributed to various proposed mechanisms of actions including Wiskott-Aldrich Syndrome protein (WASP)/neural Wiskott-Aldrich Syndrome protein (N-WASP) inhibition, Sec61 translocon inhibition, angiotensin II type 2 receptor (AT2R) inhibition, and inhibition of mTOR. The possible application of novel mycolactone analogues produced based on SAR investigations as therapeutic agents for the treatment of inflammatory disorders and inflammatory pain are discussed. Additionally, their therapeutic potential as anti-viral and anti-cancer agents have also been addressed.

Keywords: Buruli ulcer; analgesic and cytotoxic; aptamers; biomarker; immunosuppressive; mycolactone; toxin.

Figure 3

Structures of mycolactones A/B [89], C [90], D [91], E [91,92,93], E [91,92,93], F [78,94], dia-F [95,96], G [97], S1 [98], and S2 [98]. Lactone ring highlighted in red, C-linked C12–C20 side chain highlighted in blue, and polyunsaturated fatty acid side chain is in black.

Figure 5

Overview of domain and module organization of the mycolactone PKS genes (a) MlsA1 and MlsA2 from the mycolactone PKS, harbored by the plasmid pMUM001 from M. ulcerans Agy99 [111,114]; (b) subunits (MLSA1, MLSA2, and MLSB) of different domains are represented by color block [115].

Figure 1

Clinical forms of Buruli ulcer. (A) nodule, (B) plaque, (C) oedema, (D) small ulcer [9].

Figure 2

Global map showing the distribution of Buruli ulcer disease as of 2023. Data source: World Health Organization. Map production: Control of Neglected Tropical Diseases (NTD), WHO [17].

Figure 4

Complete structure of mycolactone A/B showing the core cyclic lactone ring (C1–C11) and two polyketide-derived highly unsaturated acyl side chains comprising the upper ‘Northern’ chain (C12–C20) and the longer ‘Southern’ chain (C1′–C16′). Under suitable laboratory conditions and light, mycolactone exists as geometric isomers centered around the double bond at C4′ C5′ in a 3:2 ratio.

Figure 6

Synthetic strategy employed by Kishi for the total syntheses of mycolactone A/B.

Scheme 1

Synthesis of the C1–C7 fragment. 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%.

Scheme 2

Synthesis of the C8–C13 fragment. Reagents and conditions: (1) m-CPBA, CH₂Cl₂, 0 to 20 °C, 80%; (2) propyne, THF, n-BuLi, BF₃•OEt₂, −78 °C, 94%; (3) TBAF, THF, 73%; (4) cyclopentanone, TsOH, benzene, 76%; (5) Cp₂ZrHCl, THF, 50 °C, 1 h; (6) I₂, THF, 62%.

Scheme 3

Synthesis of C14–C20 fragment. Reagents and conditions: (1) TsOH, MeOH/THF, 79%; (2) cyclopentanone, TsOH, 83%; (3) O₃, CH₂Cl₂ then PPh₃, −78 °C, 97%; (4) DAMP, ^tBuOK, THF, −78 °C, 88%; (5) n-BuLi, MeI, −78 to 20 °C, 99%; (6) Cp₂ZrHCl, THF, 50 °C then I₂, THF, 79%.

Scheme 4

Kishi’s first-generation approach toward the synthesis of the fully hydroxy-protected core structure of mycolactones [102]. Reagents and conditions: (a) t-BuLi (3 equiv), ZnCl₂, Pd(Ph₃P)₄, THF, 60%; (b) 1. CH₂Cl₂/H₂O/TFA, (8:2:0.5), 77%; 2. PivCl, pyr., 99%; 3. TESCl, imid., CH₂Cl₂, 91%; 4. DiBAl-H, CH₂Cl₂, −78 °C, 98%; 5. I₂, Ph₃P imidazole, Et₂O-MeCN (3:1), 91%; (c) t-BuLi (3 equiv), ZnCl₂, Pd(Ph₃P)₄, THF, 50%; (d) 1. HF·pyr./pyr./THF (1:1:4), THF, 72%; 2. TEMPO, NCS, Bu₄NCl, CH₂Cl₂-pH 8.6 buffer (1:1), 95%; 3. NaClO₂, NaH₂PO₄, m-(MeO)₂-C₆H₄, DMSO-t-BuOH (1:1), 94%; (e) 1. Cl₃C₆H₂COCl, i-Pr₂NEt, PhH; DMAP, PhH, 70%; 2. CH₂Cl₂/H₂O/TFA (8:2:0.5), 62%; 3. HF·pyr., MeCN, 77%; (f) 1,1-dimethoxycyclopentane—25, p-TsOH, benzene, 80%.

Scheme 5

Kishi’s second-generation approach toward the synthesis of the core structure of mycolactone. Reagents and conditions: (a) Zn, Cu(OAc)₂, Pd(PPh₃)₄, LiCl, NMP, 60 °C, 83%; (b) 1. CH₂Cl₂/H₂O/TFA (16:4:1), 90%; 2. TIPSCl, imidazole, DMF, 100%; 3. LiOH, THF/MeOH/H₂O, (4:1:1), 81%; (c) Cl₃C₆H₂COCl, i-Pr₂NEt, benzene, then DMAP, benzene 96%; (d) 1. HF•py-py-CH₃CN, 90%; 2. Ph₃P, imidazole, I₂, CH₂Cl₂, 98%; (e) Zn, Cu(OAc)₂, Pd(PPh₃)₄, LiCl, NMP, 60 °C, 80%; (f) DDQ, CH₂Cl₂/H₂O, 91%.

Scheme 6

Assembly of the mycolactone core. Reagents and conditions: (a) Zn, Cu(OAc)₂, Pd(PPh₃)₄, LiCl; (b) TFA, wet CH₂Cl₂; (c) 1. TIPSOTf, 2,6-lutidine; 2. LiOH; (d) Cl₃C₆H₂COCl, i-Pr₂NEt, DMAP (e) HF·pyr., pyr.; (f) Ph₃P, I₂ imidazole; (g) Zn, Cu(OAc)₂, Pd(PPh₃)₄, LiCl.

Figure 7

Kishi’s synthetic strategies for the synthesis of suitably protected pentaenoic acid.

Scheme 7

Synthetic route for a suitably protected pentaenoic acid 49. Reagents and conditions: (a) LDA, THF, −78 °C, rt, 1 h, 94%; (b) LiOH, THF/MeOH/H2O (4:1:1), rt, 18 h, 100%.

Scheme 8

Synthesis of the C9′–C16′ tris-TBS aldehyde 47. Reagents and conditions: (a) NaH, (EtO)₂P(O)CH₂CO₂Et, THF, rt, 1 h, 64%; (b) AD-mix-a, MeSO₂NH₂, t-BuOH/H₂O (1:1), 40 h, 0 °C, 70%; (c) 1. TBSOTf, 2,6-lutidine, CH₂Cl₂, 0 °C, 99%; 2. DIBAL, CH₂Cl₂, 89%; 3. SO_3·py, i-Pr₂NEt, CH₂Cl₂-DMSO (3:2); 4. Ph₃P=C(Me)CO₂Et, toluene, 110 °C, 83% (2 steps); (d) DIBAL, CH₂Cl₂, −78 °C, 57%; (e) SO₃·py, i-Pr₂NEt, CH₂Cl₂-DMSO (3:2) 100%.

Scheme 9

Synthesis of the C1′–C8′ phosphonate 46. Reagents and conditions: (a) 1. TBSCl, imidazole, DMF; 2. O₃, CH₂Cl₂, −78 °C, then Ph₃P; 3. Ph₃P=C(Me)CO₂Et, CH₂Cl₂; 4. DIBAL, CH₂Cl₂, −78 °C, 25% (four steps); (b) 1. SO₃·py, i-Pr₂NEt, CH₂Cl₂/DMSO (3:2); 2. Ph₃P=C(Me)CO₂Et, benzene, 90 °C, 80% (2 steps); (c) 1. DIBAL, CH₂Cl₂, −78 °C; 2. SO₃·py, i-Pr₂NEt, CH₂Cl₂/DMSO (3:2); 3. Ph3P=C(Me)CO₂Et, benzene, 90 °C, 89% (3 steps); (d) 1. TBAF, THF, 87%; 2. PBr₃, Et₂O, 77%; 3. (EtO)₃P, 90 °C, 96%.

Scheme 10

Completion of Kishi’s first-generation total synthesis of mycolactone A/B. Reagents and conditions: (a) Cl₃C₆H₂COCl, i-Pr₂NEt, DMAP, PhH, rt, 20 h, 90%; (b) 1. TBAF, THF, rt, 1 h, 81%; 2. THF/HOAc/H₂O (2:2:1), rt, 10 h, 67% with one recycle.

Figure 8

Mycolactone A/B and proposed key steps of its total synthesis.

Figure 9

Structure of truncated and biotinylated derivative of mycolactone synthetic (PG-204) for the detection of mAbs.

Figure 10

Schematic illustration of Kishi’s improved mycolactone TLC detection method exploiting derivatization with 2-napthylboronic acid as a fluorescence enhancer [167].

Figure 11

Proposed molecular targets and mechanisms of action for mycolactone-mediated ulcerative, immunosuppressive, and analgesic properties [58].

Figure 12

Structure of mycolactone A/B and its synthetic analogues 56a and 56b with extended polyketide southern side chain.

Figure 13

Synthetic analogues by Altmann and Pluschke.

Figure 14

Blanchard synthetic analogues.