University of Groningen Total synthesis of mycolic acids and site-selective functionalization of aminoglycoside antibiotics Tahiri, Nabil

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University of Groningen

Total synthesis of mycolic acids and site-selective functionalization of aminoglycoside

antibiotics

Tahiri, Nabil

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Tahiri, N. (2019). Total synthesis of mycolic acids and site-selective functionalization of aminoglycoside antibiotics. University of Groningen.

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531024-L-sub01-bw-Tahiri 531024-L-sub01-bw-Tahiri 531024-L-sub01-bw-Tahiri 531024-L-sub01-bw-Tahiri Processed on: 3-5-2019 Processed on: 3-5-2019 Processed on: 3-5-2019

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Chapter 6:

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6.1 Summary

Tuberculosis (TB) is, up until today, still the most lethal bacterial disease known to humans. It is estimated that roughly a quarter of the world population is infected, either by a latent or active form. This number continues to rise every year, as is demonstrated by the estimated 10 mil new infections in 2017 alone.[1]_{When diagnosed in time, TB}

can be treated by subjecting the patient to a dedicated antibiotic regimen lasting for several months. Unfortunately, these antibiotics are losing their effectiveness, as multi-drug resistant tuberculosis (MDR-TB) and extensively multi-multi-drug resistant tuberculosis (XDR-TB) are on the rise. Treatment of these forms requires the administration of larger quantities of more toxic drugs over an extended period, ranging from six months up to two years, and can severely affect the patients’ quality of life. Part of this difficulty in the treatment can be attributed to M. tuberculosis’ unusually fortified cell envelope. The outer membrane of the cell envelope, also referred to as the mycomembrane, consists of a high abundance of mycolic acids. These outer cell wall components have been shown to be able to fold within the cell wall,[2]_{thereby forming a}

very dense and almost impenetrable layer of lipids. Mycolic acids are essential for

Mycobacteria’s survival, and are categorized in three classes: α-, methoxy-, and

ketomycolic acids. These highly lipophilic fatty acids are isolated as a mixture of homologues, and range from 74 to 88 carbon atoms, depending on the class.

Mycolic acids are biologically very relevant, as they induce part of the immune response upon infection, and are therefore intensively studied in biology. Although they have been known for over a century, a long standing and unsolved problem is the elucidation of their stereochemical configuration. The tuberculosis field is developing vaccine candidates and diagnostic kits based on mycolic acids, but is reluctant doing so based on natural material, because of potential contamination and the cumbersome culturing of pathogenic M. tuberculosis. Therefore, the tuberculosis field would gain considerable progress with the complete resolution of the stereochemistry, and a high yielding asymmetric total synthesis of mycolic acids. In the work described in this dissertation, I attempted to approach TB research from two different angles. On one hand, I synthesized four methoxymycolic acid diastereomers, which allow for biological studies, and should function as reference material in (future) stereochemical studies. On the other hand, I attempted the synthesis of novel aminoglycoside antibiotics, which potentially could lead to useful antibiotics. At the center stage in the research conducted towards aminoglycosides was the regioselective oxidation of saccharides, developed by the Minnaard group in 2013.[3]

Our retrosynthetic approach in the synthesis of the four methoxymycolic acid diastereomers is shown in Scheme 1. We disconnected the molecule in three fragments of equal stereochemical complexity, of which the gram-scale asymmetric synthesis is described in chapter 2. In order to prepare four diastereomers, in which we vary the absolute stereochemistry in the syn α-methyl methoxy (fragment C) and the cis

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cyclopropyl moieties (fragment B), we prepared both enantiomers of these fragments, while maintaining a R,R stereochemistry in fragment A. Fragments A, B, and C were obtained in nine, eight, and eight steps in the longest linear sequence, and in 24%, 38%, and 45% yield, respectively. In chapter 3 these fragments were combined, which required eight additional steps to arrive at the mycolic acid as depicted in Scheme 1. The four diastereomers could be obtained in 17 steps each in the longest linear sequence, and in 12% overall yield. The obtained mycolic acids were derivatized as the (R)-, and (S)-glycerol monomycolates (GroMM), and glucose monomycolates (GMM). Biological tests of the free mycolic acids indicated that the diastereomers with S,S stereochemistry at the α-methyl methoxy moiety (Fragment C in Scheme 1) possess a significantly better activation of T cells over the R,R stereochemistry at this position. On the other hand, the differences between diastereomers with opposite stereochemistry in the cyclopropyl ring was less pronounces in free MAs, but isomers with R,S stereochemistry (stereochemistry as is depicted in the full molecule in Scheme 1) resulted in higher T cell activation over their S,R counterparts (with cyclopropyl substituents pointed to the back).

The obtained optical rotations of the methyl esters were in agreement with those obtained by Al Dulayymi et al.[4]_{and Asselineau et al.}[5]_{These values were used to}

determine the specific molar rotations, which were in line with the expected values based on the principle of optical superposition. By preparing both R,R diastereomers in the syn α-methyl methoxy moiety, it was demonstrated for the first time that this is a valid strategy for mycolic acids. It is safe to assume that the remotely located stereocenters in methoxymycolic acid do not influence each other’s specific rotation, which reinforces the S,S stereochemistry at the α-methyl methoxy segment as proposed by Asselineau et al.[5]

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The remainder of this dissertation focused on the application of the regioselective palladium catalyzed oxidation of (amino) saccharides. In chapter 4 we focused first on improving the TON of the palladium catalyst 1 in the regioselective oxidation under

aerobic conditions. This oxidation can be executed using either benzoquinone or air as

the terminal oxidant. The application of the former results in fast reaction rates and a good TON, but is accompanied with the formation of stoichiometric amounts of hydroquinone, which increases the difficulty of product isolation. On the other hand, the use of oxygen results in clean reaction products, but is accompanied with the generation of highly reactive palladium hydroperoxide species, originating from the partial reduction of oxygen by the catalyst. As a result, aerobic oxidations with 1 require high catalyst loadings of 10 mol% due to inactivation of the catalyst via autoxidation of 1 resulting in catalytically inactive 4 (Scheme 2). We realized that by deuteration of both methyl substituents, we could provide an effective strategy towards enhanced catalyst stability under aerobic conditions. The reduction of catalyst degradation allowed for the aerobic oxidation of carbohydrates using acceptable catalyst loadings (3 mol% of dimer

1-d6). The straightforward deuteration of the methyl substituents in neocuproine allowed

for an increase in the turnover number in the aerobic alcohol oxidation of 2-heptanol with at least 1.6 times and for methyl glucoside with 1.8 times.[6]

Scheme 2. The regioselective aerobic oxidation of methyl D-D-glucopyranoside.

In chapter 5 we used catalyst 1 in the regioselective oxidation of N-protected neomycin B. The oxidation product was used in the synthesis of a library of novel amphiphilic aminoglycosides. Initially, we planned to install the lipophilic group via either a reductive amination or oxime ligation. After optimization of the reaction conditions we obtained a library of amphiphilic aminoglycosides, in which the lipophilic chain consisted of 8 up to 18 carbon atoms, and was installed via an oxime bond (Scheme 3). Although the E/Z isomers could be partially separated on semi-prep HPLC, we demonstrated that in aqueous solutions at room temperature isomerization towards an equilibrium takes place. A preliminary viability assay on B. subtilis did not show significant difference between E/Z isomers, but did show better activity for the C12, C14, and C16 over the C8, C10 and C18 tails. Future test will need to be conducted to test antifungal activity and antibacterial activity on more relevant strains.

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Scheme 3. The concise synthesis of an amphiphilic neomycin B library.

6.2 Work in progress, and future perspectives

In this section the work in progress along with future perspectives are described. In section 6.2.1 the work regarding the stereochemical determination of mycolic acids is described, and section 6.2.2 describes the research related to the development of novel aminoglycoside antibiotics (AGAs).

6.2.1 Mycolic acid research

Although we managed to synthesize sufficient amounts of material to aid in biological studies, the structural elucidation studies towards the cyclopropyl ring’s absolute configuration have not yet been performed. Because of the long distance between the functional groups, NMR is not of great help as the spectra of the synthetic compounds turned out to be superimposable. Therefore, other tools such as HPLC will be necessary to distinguish between the diastereomers. This is complicated by the high lipophilicity of the compounds that complicates finding conditions under which the diastereomers dissolve and can be separated. Furthermore, the natural compound is isolated as a mixture of homologues. These natural methoxy MAs possess different chain lengths, and separation of the compounds by prep-HPLC will probably be necessary, in order to allow for direct comparison with the synthetic material. To the best of our knowledge, the separation of methoxymycolic acid homologues has not been reported. However, Schnoes and Takayama reported the analytical separation of α-mycolic acid homologues by HPLC in 1978.[7]_{Their method could form a good starting point to}

develop our own preparative method for the separation of methoxymycolic acid homologues.

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The installation of a UV-active group aids in the detection of the mycolic acids, which inherently allows detection of smaller quantities compared to ELSD. Therefore, we installed a phenylacyl ester on the carboxylic acid moiety, by using a slightly modified procedure published by Durst et al. in 1975.[8]_{Alkylation of the 4 mycolic acids (at 10}

mg scale) with α,p-dibromoacetophenone in the presence of stoichiometric amounts of crown ether (Scheme 4) resulted in the mycolic acid derivatives 10a-d in good yields and purity after column chromatography. At this moment, the chromatographic separation of these derivatives is being studied.

Scheme 4. Synthesis of the mycolic p-bromophenylacyl esters. Although only one diastereomer is

shown, all four individual diastereomers were prepared.

Clearly, the high lipophilicity of these molecules does not contribute to the convenience in our structure elucidation studies. Reducing the amount of lipophilic bulk could possibly have a positive influence on the chromatographic separation of the different isomers. Because of the limited number of functional groups within the molecule, the possibilities are unfortunately very limited. A common used strategy in pyrolysis gas chromatography is the retro-aldol reaction of the trimethylsilyl ether derivatives of the methyl esters, resulting in the C20-C26 methyl ester and the meroaldehyde. Both can be detected under the correct conditions, but resolutions are usually not great.[9,10]_Although

efficient under GC conditions, extending this concept towards a mg scale laboratory setup in order to obtain mg quantities of meroaldehyde is probably difficult due to the extremely high temperatures of 350 °C. Moreover, cleavage under these conditions is unselective and results in a complex mixture of cleavage products (in GC) that would need to be separated.[9]_{However, when we attempted the Mitsunobu reaction of free}

mycolic acids with solketal in chapter 3 (Scheme 5A), we obtained olefin 11 in which both the alcohol and carboxylic acid functionalities were removed. I propose that this olefin can be subject to ozonolysis in a second step followed by a reductive workup to result in alcohol 12 (Scheme 5B). If this degradation would be performed on all 4 synthetic diastereomers, one would obtain the complete set of four stereoisomers. With HPLC we would possibly be able to differentiate between the diastereomers. Fortunately, because the stereochemistry at the α-methyl methoxy segment is known (S,S), we can limit the separation challenge to two diastereomers (the S,S – R,S and the

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Scheme 5. A) The undesired product in the Mitsunobu reaction of β-hydroxy unprotected MA

(Chapter 3). B) possible degradation experiments to aid in the structure elucidation of the cyclopropyl ring. C) The undesired product in the RuO4 oxidation of sulfone 15 (Chapter 3).

In addition, the lipophilicity could potentially be reduced even further to maintain only the cyclopropyl in the molecule. This would eventually result in 1 of the 2 possible enantiomers if the natural sample is used, and therefore chromatographic separation would require chiral HPLC. We noticed in the oxidation of sulfide 15 (Chapter 3) with too high loadings of RuO4 that the oxidation to the corresponding sulfone was possible,

but concomitant oxidation of the methoxy moiety to the ketone was hard to suppress (Scheme 6a). Therefore, RuO4 oxidation of olefin 11 could result in the keto acid. A

subsequent Baeyer-Villiger oxidation followed by hydrolysis of the newly formed ester should result in 13, which contains only the cis cyclopropyl ring, flanked by two long aliphatic carboxylic acids on both ends. However, because the only difference in both cyclopropyl substituents would be only 1 extra carbon in the right hand over the left hand segment in 13, we could probably run into trouble in the separation of the enantiomers. Fortunately, this problem can be easily solved by derivatization of only one of the two functional groups in 13 or 14. Although this strategy will require 4 steps to arrive at 14 (more if only 1 alcohol is derivatized into a UV active group), we have enough synthetic material available and the extraction of natural samples can be performed to yield sufficient amounts. In addition, applying this strategy on natural mycolic acid has besides significant reduction of the lipophilicity an additional important advantage. By removing the complete α-branch and a significant part of the

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meromycolic chain, separation of homologues should be easier since the number of possible homologous should be reduced in 14 compared to methoxymycolic acid. An alternative method for the determination of the cyclopropyls absolute stereochemistry could be the application of X-ray analysis on the methoxymycolic acids, or the degradation products thereof like 14, using the crystalline sponge technique developed by the group of Fujita in 2013.[11]_{Microgram amounts of sample of unknown}

stereochemistry can be absorbed into metal-organic frameworks (MOFS) and subsequently analyzed by X-ray. This technique solves the intrinsic limitation of X-ray diffraction analysis, namely the requirement for crystalline samples. In order to demonstrate the power of their technique, the authors were able to determine the remotely isolated methyl branch in 17 (Figure 1). This highly flexible molecule consists of a C28 hydrocarbon chain with a methyl branch that is located just slightly off-center. Both the left- and the right-hand side of the molecule show great similarities. They both contain a terminal alkyne flanked by an allylic alcohol with the same stereochemistry. The only difference is that the left-hand side in 17 (as depicted in Figure 1) is one carbon shorter compared to the right-hand side, which is the exact same situation as in

14. However, because 17 already contained a chiral center of known stereochemistry,

the elucidation of the stereochemistry at C14 was significantly relieved. Although it would be still possible to solve the absolute stereochemistry in 14 using the chiral sponge technique, since the MOFs contain a heavy atom, derivatization of one or both primary alcohols in 14 with, for example a chiral amino acid of known stereochemistry would simplify this task.

Figure 1. The structure of miyakosyne A (17), which was subjected to the crystal sponge

technique by Fujita and coworkers. The stereochemistry of the methyl at position 14 was determined to be of S stereochemistry.

6.2.2 Development of novel AGAs for targeting APH(3’)

The discovery of streptomycin, the very first aminoglycoside antibiotic (AGA) discovered by Waksman in 1943,[12]_{triggered the isolation and later the design of many}

(semi-) synthetic AGAs. Although AGAs are known for their possible side-effects,[13]

some of them still hold significant clinical relevancy, and are still listed by WHO as essential medicines. For example, streptomycin, the first effective antibiotic against Tuberculosis (TB), has been recommended as a first-line drug against TB by WHO until 2017, after which it has been moved to the list of second-line drugs, where it still is today.[14,15]_{Moreover, the administration alongside other antibiotics such as β-lactams}

or vancomycin, has been demonstrated to assert synergistic effects, resulting in an enhanced effectiveness against gram-positive bacteria.

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Unfortunately, the rising antibiotic resistance has significantly decreased the effectiveness of many antibiotics, including those belonging to the AGA class. The general main resistance mechanisms by which antibiotics are disabled by bacteria are: decreased drug accumulation in the pathogen via decreased absorption and increased efflux of the drug, alteration of the target site, alteration of the metabolic pathway, and modification of the antibacterial drug. Of these mechanisms, the latter is the most prevalent for AGAs in clinical settings. Consequently, the aminoglycosides are modified by aminoglycoside modifying enzymes (AMEs) produced by resistant pathogens, at positions which are essential for binding to the bacterial ribosome. These AMEs are categorized by the modification they install, which is N-acetylation for acetyltransferases (AACs), phosphorylation for phosphotransferases (APHs), and O-nucleotidylation for nucleotidyltransferases (ANTs). Depending on the strain, modification can happen at one or more sites within the administered aminoglycoside. As can been deduced from Scheme 6, the number of AMEs that are effective on kanamycin B is strikingly high. Modification can occur at any position throughout ring I and II, with the exception of position 5 in ring II. Coordination of aminoglycosides with the bacterial ribosome is mainly driven by favorable electrostatic interactions between the positively charged aminoglycoside and the negatively charged RNA. The introduction of a negative charge on the aminoglycoside at O3’ in the form of a phosphate by APH(3’) destabilizes this interaction and prevents the aminoglycoside from coordinating with the RNA (Scheme 6).

Scheme 6. The identified AMEs (depicted in red) which target kanamycin B. The product of

phosphorylation by APH(3’) is shown in the Scheme.

The most frequently applied strategy to overcome the structural alteration of AGAs by these resistance enzymes, is the chemical modification of the existing natural AGAs at the specific positions that are susceptible to enzymatic modification. However, subtle modification can have a large impact on the antibiotic activity, and it is hard to predict how these minor modifications made by chemists influence the activity of the novel antibiotic. For example, methylation of O3’ in kanamycin (compound 18 in Figure 2),[16]_{in order to overcome deactivation by APH(3’), resulted in an inactive antibiotic,}

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resulted in excellent activities against the wild type. Moreover, pathogenic strains expressing the APH(3’) enzyme were susceptible to this kanamycin derivative.

Figure 2. Methylated (18) and deoxygenated (19) kanamycin derivatives.

Other less straightforward, but very elegant approaches include kanamycin derivatives

20[18]_{and 23,}[19]_{both developed by the Mobashery research group (Scheme 7). These}

kanamycin derivatives are cleverly designed, so that they are not affected by phosphorylation. The keto functionality in compound 20 mainly resides as the hydrate under physiological conditions. Phosphorylation of the hydrate, results in decomposition of the phosphate, regenerating the keto derivative 20. In the second example, phosphorylation of 23 by APH(3’) results, after elimination of the phosphate, in α,β-unsaturated nitro compound 25. This highly reactive product is then, while still in close proximity to the active site, trapped by a nucleophilic amino acid residue resulting in deactivation of the enzyme. Although these examples hold great promise as potential candidates, their application beyond proof of principle is limited by their lengthy synthesis and high costs.

Scheme 7. A) a self-regenerating kanamycin derivative. B) a mechanism-based inhibitor of

APH(3’). R represents ring I and III as depicted in Figure 1.

The regioselective oxidation developed in our group,[3]_{would allow quick access to a}

range of interesting molecules with just a minimal number of steps. We have shown earlier that this methodology is applicable on N-protected aminoglycosides such as kanamycin, neomycin B and amikacin. The N-protected oxidation product of these aminoglycosides (like 7) should serve as a key intermediate in the synthesis of novel

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potential AGAs. For example, the ketone could be removed via the reduction of the corresponding tosylhydrazone, yielding a 3’-deoxygenated aminoglycoside, which should not be susceptible to phosphorylation at the 3’ position anymore (as is the case for 19). However, since both alcohols at the 5’’ and 3’ positions (see Scheme 3 for numbering) can be phosphorolated by the APH(3’) enzyme in 4,5-linked aminoglycosides (e.g neomycin and paromomycin),[20]_{both alcohols need to be}

removed or modified. In analogy to 23, an epoxide moiety could aid as an electrophilic trap for the APH(3’) enzyme. Epoxide formation of the ketone could be achieved in a single step with diazomethane, or alternatively with a Corey-Chaykovsky reaction. Furthermore, the introduction of a tertiary alcohol at this position could result in increased steric hindrance at this position, which could slow down the rate of phosphorylation by APH(3’).

Preliminary model studies on 26 showed that epoxide formation, and indium mediated allyl addition on the ketone is possible, and resulted in the products as single diastereomers (Scheme 8). Furthermore, transformation of the keto-sugar to the corresponding tosylhydrazone, followed by a two-step reduction procedure,[21]_resulted

in the 3-deoxy sugar in 3 steps from the ketone.

Scheme 8. Deoxygenation, allylation, and epoxide formation model studies on keto sugar 26.

6.3 Experimental

General remarks

All moisture sensitive reactions were performed using flame-dried glassware under nitrogen atmosphere using standard Schlenk techniques and dry solvents. Reaction temperatures below 0 °C refer to internal temperatures, while reaction temperatures higher than rt refer to heating bath temperatures. Dry solvents were taken from a MBraun solvent purification system (SPS-800). All other reagents were purchased from Sigma-Aldrich, Acros, TCI Europe, Strem chemicals or Fluorochem and used without further purification unless noted otherwise.

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TLC analysis was performed with Merck silica gel 60/Kieselguhr F245, 0.25 mm. Compounds were visualized using either a KMnO4 stain (K2CO3 (40 g), KMnO4 (6 g),

water (600 ml) and 10% NaOH (5 ml)), anisaldehyde stain (EtOH (135 ml), H2SO4 (5

ml), AcOH (1.5 ml), p-anisaldehyde (3.7 ml)), PMA stain (phosphomolybdic acid (10 g) in ethanol (100 ml)) or elemental iodine.

Flash chromatography was performed using SiliCycle silica gel type SiliaFlash P60 (230-400 mesh) as obtained from Screening Devices.

1_{H- and}13_{C-NMR spectra were recorded on a Agilent MR400 (400 and 100 MHz,}

respectively) or a Bruker Avance NEO 600 (600 and 150 MHz, respectively). CDCl3

was used as solvent unless stated otherwise. Chemical shift values are reported in ppm with the solvent resonance as the internal standard (CDCl3: δ7.26 for 1H, δ77.16 for 13_{C). Data are reported as follows: chemical shifts, multiplicity (s = singlet, d = doublet,}

dd = double doublet, ddd = double double doublet, dt = double triplte, td = triple doublet, t = triplet, q = quartet, p = pentet, b = broad, m = multiplet), coupling constants

J (Hz), and integration.

Experimental procedures

General procedure for the esterification of methoxymycolic acids as p-bromophenylacyl esters:

To a solution of the mycolic acid in CHCl3 (0.027 M) was added K2CO3 (2.2 equiv.)

followed by a mixture of 18-crown-6 (1.0 equiv.), and 2,4’-dibromoacetophenone (1.5 equiv.) in acetonitrile (0.07 M in 18-crown-6) at rt. The mixture was stirred at 85 °C during 45 min. After TLC (2.5% MeOH in DCM) indicated complete consumption of the mycolic acid, the reaction was allowed to cool to rt, and diluted with pentane (1 ml). Celite (80 mg) was added, and the mixture was evaporated in vacuo. The residue was purified by flash column chromatography (5% EtOAc in pentane), yielding a colorless oil which solidified upon standing.

Compound 10a:

Methoxymycolic acid (9.2 mg, 1.0 equiv.), K2CO3 (2.3 mg, 2.2 equiv.),

18-crown-6 (2.0 mg, 1.0 equiv.) and 2,4’-dibromoacetophenone (2.9 mg, 1.5 equiv.) were reacted according to the aforementioned general procedure, using 300 μl and 100 μl of CHCl3 and acetonitrile, respectively. The reaction afforded the product as

a white solid (8.9 mg, 84% yield) after column chromatography.

1_{H-NMR (400 MHz, Chloroform-d) δ 7.81 – 7.75 (m, 2H), 7.67 – 7.61 (m, 2H), 5.47}

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171 3H), 3.24 (d, J = 6.9 Hz, 1H), 2.95 (dt, J = 7.1, 4.1 Hz, 1H), 2.62 – 2.54 (m, 1H), 1.82 – 1.68 (m, 1H), 1.68 – 0.99 (m, 146H), 0.94 – 0.79 (m, 9H), 0.69 – 0.60 (m, 2H), 0.59 – 0.51 (m, 1H), -0.34 (td, J = 5.2, 3.9 Hz, 1H). 13_{C-NMR (151 MHz, CDCl} 3) δ 192.15, 174.39, 132.72, 132.47, 129.67, 129.49, 85.59, 72.81, 65.72, 57.87, 52.53, 35.48, 35.19, 32.52, 32.09, 30.64, 30.39, 30.14, 30.10, 29.86, 29.82, 29.77, 29.75, 29.70, 29.60, 29.52, 29.37, 28.88, 27.74, 27.64, 26.32, 25.83, 22.85, 15.94, 15.05, 14.28, 11.07.

HRMS (ESI) Calcd. for C93H174O5 ([M + H]+): 1450.25 100%), 1451.26 (100%),

1452.25 (100%), 1453.25 (95%), found: 1450.25 (100%), 1451.26 (100%), 1452.25 (95%), 1453.26 (95%).

Compound 10b:

a white solid (8.3 mg, 76% yield) after column chromatography.

1_{H-NMR (400 MHz, CDCl} 3) δ 7.81 – 7.76 (m, 2H), 7.68 – 7.62 (m, 2H), 5.47 (d, J = 16.4 Hz, 1H), 5.27 (d, J = 16.5 Hz, 1H), 3.81 – 3.72 (m, 1H), 3.34 (s, 3H), 3.24 (d, J = 6.9 Hz, 1H), 2.98 – 2.92 (m, 1H), 2.62 – 2.55 (m, 1H), 1.81 – 1.69 (m, 1H), 1.69 – 1.00 (m, 146H), 0.94 – 0.79 (m, 9H), 0.69 – 0.60 (m, 2H), 0.59 – 0.52 (m, 1H), -0.34 (td, J = 5.3, 4.0 Hz, 1H).13_{C-NMR δ 192.14, 174.38, 132.72, 132.47, 129.67, 129.49, 85.59,} 72.81, 65.72, 57.87, 52.53, 35.48, 35.19, 32.52, 32.09, 30.64, 30.39, 30.14, 30.10, 29.86, 29.82, 29.77, 29.75, 29.71, 29.60, 29.52, 29.37, 28.88, 27.74, 27.64, 26.32, 25.83, 22.85, 15.94, 15.05, 14.28, 11.07. HRMS (ESI) Calcd. for C93H174O5 ([M +

H]+_{): 1450.25 100%), 1451.26 (100%), 1452.25 (100%), 1453.25 (95%), found:}

1450.25 (100%), 1451.26 (100%), 1452.25 (95%), 1453.26 (95%).

Compound 10c:

1_{H-NMR (400 MHz, CDCl}

3) δ 7.81 – 7.75 (m, 2H), 7.67 – 7.62 (m, 2H), 5.47 (d, J =

16.4 Hz, 1H), 5.27 (d, J = 16.5 Hz, 1H), 3.81 – 3.71 (m, 1H), 3.34 (s, 3H), 3.24 (d, J = 6.9 Hz, 1H), 3.00 – 2.91 (m, 1H), 2.62 – 2.54 (m, 1H), 1.82 – 1.69 (m, 1H), 1.69 – 0.99

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172 (m, 146H), 0.95 – 0.78 (m, 9H), 0.69 – 0.59 (m, 2H), 0.59 – 0.52 (m, 1H), -0.34 (td, J = 5.3, 4.0 Hz, 1H). 13_{C-NMR (151 MHz, CDCl} 3) δ 192.15, 174.39, 132.72, 132.47, 129.67, 129.49, 85.59, 72.81, 65.72, 57.87, 52.53, 35.48, 35.19, 32.52, 32.09, 30.64, 30.39, 30.14, 30.10, 29.86, 29.82, 29.77, 29.75, 29.71, 29.60, 29.52, 29.37, 28.88, 27.74, 27.64, 26.32, 25.83, 22.85, 15.94, 15.05, 14.28, 11.07. HRMS (ESI) Calcd. for C93H174O5 ([M + H]+): 1450.25 100%), 1451.26 (100%), 1452.25 (100%), 1453.25

(95%), found: 1450.25 (100%), 1451.26 (100%), 1452.25 (95%), 1453.26 (95%).

Compound 10d:

1_{H-NMR (400 MHz, Chloroform-d) δ 7.81 – 7.76 (m, 2H), 7.67 – 7.62 (m, 2H), 5.47} (d, J = 16.4 Hz, 1H), 5.27 (d, J = 16.5 Hz, 1H), 3.81 – 3.71 (m, 1H), 3.34 (s, 3H), 3.24 (d, J = 6.9 Hz, 1H), 2.99 – 2.91 (m, 1H), 2.62 – 2.54 (m, 1H), 1.81 – 1.69 (m, 1H), 1.67 – 1.01 (m, 146H), 0.94 – 0.79 (m, 9H), 0.69 – 0.60 (m, 2H), 0.59 – 0.52 (m, 1H), -0.34 (td, J = 5.3, 3.9 Hz, 1H). 13_{C-NMR (151 MHz, CDCl} 3) δ 192.15, 174.39, 132.72, 132.47, 129.67, 129.49, 85.59, 72.81, 65.72, 57.87, 52.53, 35.48, 35.19, 32.52, 32.09, 30.64, 30.39, 30.14, 30.10, 29.87, 29.82, 29.77, 29.75, 29.71, 29.60, 29.52, 29.37, 28.88, 27.74, 27.64, 26.32, 25.83, 22.85, 15.94, 15.05, 14.28, 11.07. HRMS (ESI) Calcd. for C93H174O5 ([M + H]+): 1450.25 (100%), 1451.26 (100%), 1452.25 (100%), 1453.25 (95%), found: 1450.25 (100%), 1451.26 (100%), 1452.25 (95%), 1453.26 (95%). Deoxygenation of 26:

To a solution of ketone 26[3]_{(140 mg, 0.73 mmol, 1.0 equiv.) dissolved in MeOH (1.4}

ml) was added tosylhydrazine (150 mg, 0.80 mmol, 1.1 equiv), and the reaction mixture was stirred at 60 °C overnight. Then, the crude was absorbed on celite and purified by flask column chromatography, yielding the product (191 mg, 0.53 mmol, 73%) as a mixture of E/Z isomers in a 1:2 ratio as an off-white foam.

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To a solution of tosylhydrazone 31 (170 mg, 0.47 mmol, 1.0 equiv.) in degassed MeOH/THF (1:1, 4 ml) at rt was added a trace of methyl orange, resulting in a yellow colored solution. Then, NaCNBH3 (59 mg, 0.94 mmol, 2.0 equiv.) was added in 1

portion, and 1 M HCl in methanol (prepared by adding 341 μl AcCl in 25 ml deoxygenated MeOH) was added until the reaction reached the orange transition point (pH 3.8). The color of the solution was maintained at this point by occasional addition of more 1 M HCl when necessary during 30 min. Then, another portion of NaCNBH3

(59 mg, 0.94 mmol, 2.0 equiv.) was added, after which directly more 1 M HCl was added to re-establish the orange transition point. After another 30 min stirring at rt, TLC (7% MeOH in DCM, very minor separation between starting material and product) indicated full conversion of the hydrazine. The reaction was concentrated in vacuo, and the residue was dissolved in degassed EtOH (2.5 ml). Then, NaOAc (321 mg, 2.36 mmol, 5.0 equiv.) was added, and the reaction mixture was stirred under reflux during 30 min. The reaction mixture was concentrated in vacuo, and purified by flash column chromatography (10-15% MeOH in DCM), yielding a colorless oil (117 mg). The columned product was dissolved in MeOH (10 ml), and amberlite H+ _{resin was added,}

which resulted in gas formation. The solution was filtered, concentrated in vacuo, and coevaporated with toluene (2x), and methanol (3x), which yielded the product (29) as a colorless oil (42 mg, 0.24 mmol, 51% over 2 steps).

1_{H-NMR δ 4.57 (d, J = 3.5 Hz, 1H), 3.80 (dd, J = 11.8, 2.5 Hz, 1H), 3.69 – 3.59 (m,}

2H), 3.55 – 3.36 (m, 5H), 2.10 – 2.00 (m, 1H), 1.76 (q, J = 11.5 Hz, 1H).

Note: When the crude of the NaOAc promoted decomposition reaction was treated with Amberlite H+ _{before column} chromatography, a significant part of 29 was obtained as product 32, originating from the acid catalyzed esterification of the 6-OH and tosylsulfinic acid.

Indium mediated allylation of 26:

To a vigorously stirred suspension of indium (60 mg, 0.5 mmol, 1.0 equiv.) in deionized water (4.5 ml) at rt was added 26[3]_{(100 mg, 0.5 mmol 1.0 equiv.), followed by}

allylbromide (68 μl, 0.8 mmol, 1.5 equiv.) in THF (0.5 ml). After vigorous stirring overnight at rt, 1_{H-NMR showed full and extremely clean conversion to a single}

diastereomer. The product was evaporated in vacuo, yielding the product as a single diastereomer (130 mg, 0.6 mmol, 106%) as a gray solid, which most probably still contained indium salts. No further action to remove the salts was undertaken.

1_{H-NMR (400 MHz, MeOD) δ 5.89 – 5.76 (m, 1H), 5.23 – 5.07 (m, 2H), 4.70 (d, J =}

3.9 Hz, 1H), 3.87 – 3.79 (m, 1H), 3.74 – 3.62 (m, 2H), 3.47 (d, J = 4.0 Hz, 1H), 3.44 (s, 3H), 3.40 – 3.34 (m, 1H), 2.54 – 2.39 (m, 2H).

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Epoxide formation:

To a solution of trimethylsulfoxonium iodide (253 mg, 1.15 mmol, 1.15 equiv.) in dry DMSO (2 ml) was added NaH (60% dispersion in mineral oil, 41 mg, 1.05 mmol, 1.05 equiv.) in a single portion. The reaction mixture was stirred at rt during 30 min, after which a solution of 26[3]_{(190 mg, 1.0 mmol, 1.0 equiv.) in dry DMSO (1 ml) was}

added. The reaction mixture was stirred at 40 °C during 2.5 h, after which most of the DMSO was removed by vacuum distillation at 50 °C. The crude was purified by charcoal column chromatography[22]_{(100% H}

2O, 5CV, followed by a 2-10% EtOH in

H2O gradient) yielding the product as a colorless oil (48 mg, 23%) The product still

contained 23 mol% DMSO according to NMR.

1_{H-NMR (400 MHz, CD} 3OD) δ 4.74 (d, J = 3.9 Hz, 1H), 3.89 (d, J = 3.9 Hz, 1H), 3.83 (dd, J = 11.5, 1.9 Hz, 1H), 3.78 (d, J = 9.7 Hz, 1H), 3.74 (dd, J = 11.5 Hz, 5.2 Hz, 1H), 3.72 – 3.65 (m, 1H), 3.43 (s, 3H), 2.85 – 2.79 (m, 2H). 13_{C-NMR (101 MHz, CD} 3OD) δ 100.99, 72.14, 66.85, 64.72, 62.67, 60.77, 55.74, 42.89.

6.4 References

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Witte, A. J. Minnaard, Chem. Commun. 2016, 52, 2189–2191. [7] H. K. Schnoes, K. Takayama, J. Liq. Chromatogr. 1981, 4, 1207–1218.

[8] H. D. Durst, M. Milano, E. J. Kikta, S. A. Connelly, E. Grushka, Anal. Chem. 1975, 47, 1797–1801.

[9] K. Kaneda, S. Naito, S. Imaizumi, I. Yano, S. Mizuno, I. Tomiyasu, T. Baba, E. Kusunose, M. Kusunose, J. Clin. Microbiol. 1986, 24, 1060–1070.

[10] S. Toriyama, I. Yano, M. Masui, M. Kusunose, E. Kunose, FEBS Lett. 1978, 95, 111– 115.

[11] Y. Inokuma, S. Yoshioka, J. Ariyoshi, T. Arai, Y. Hitora, K. Takada, S. Matsunaga, K. Rissanen, M. Fujita, Nature 2013, 495, 461–466.

[12] A. Schartz, E. Bugie, S. A. Waksman, Exp. Biol. Med. 1944, 55, 66–69. [13] A. Prayle, A. Watson, H. Fortnum, A. Smyth, Thorax 2010, 65, 654–658.

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Guidelines for Drug- Resistant Tuberculosis 2016, 2016.

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[17] S. Umezawa, Y. Nishimura, H. Hineno, K. Watanabe, S. Koike, T. Tsuchiya, H. Umezawa, Bull. Chem. Soc. Jpn. 1972, 45, 2847–2851.

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