Total Synthesis of Complex Biosynthetic Late ... - ACS Publications

Subscriber access provided by University of Sunderland

Note

Total Synthesis of Complex Biosynthetic Late-Stage Intermediates and Bioconversion by a Tailoring Enzyme from Jerangolid Biosynthesis Frederick Lindner, Steffen Friedrich, and Frank Hahn J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.8b02047 • Publication Date (Web): 04 Oct 2018 Downloaded from http://pubs.acs.org on October 4, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 13 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

The Journal of Organic Chemistry

Total Synthesis of Complex Biosynthetic Late-Stage Intermediates and Bioconversion by a Tailoring Enzyme from Jerangolid Biosynthesis Frederick Lindner, Steffen Friedrich†, Frank Hahn* Professur für Organische Chemie (Lebensmittelchemie), Department of Chemistry, Fakultät für Biologie, Chemie und Geowissenschaften, Universität Bayreuth, Universitätsstraße 30, 95447 Bayreuth (Germany)

Supporting Information Placeholder ABSTRACT: A highly convergent access to the late-stage biosynthetic intermediates projerangolid and jerangolid E is presented and its utility demonstrated by the synthesis of novel non-natural jerangolid derivatives. The key steps are fragment couplings by Julia-Kocienski olefination and Olefin cross metathesis as well as a stereoselective tetrahydropyran formation by intramolecular oxaMichael addition. Bioconversion experiments with the tailoring Omethyltransferase JerF confirmed its proposed biosynthetic role and revealed relaxed substrate specificity of this enzyme as well as tolerance to organic co-solvents.

The jerangolids (1) are myxobacterial polyketides from Sorrangium cellulosum So ce307 with promising antifungal activity and exceptional low toxicity in mammals.1 They are closely related to the ambruticins (2) with which they share the assumed pharmacophoric unit consisting of a dihydropyran (DHP) and a skipped diene (Figure 1a). Beside pyrrolnitrin, they are the only known compounds, which target the high osmolarity glycerol (HOG) signaling pathway.2–6 Total syntheses of natural jerangolids and ambruticins are described in the literature and semisynthetic efforts gave rise to ambruticin libraries with variations in the western part of the molecule.7–12 No access to libraries that would allow investigating the effect of alterations in the pharmacophore are yet reported. In agreement with the structural identities, the biosynthetic pathways of 1 and 2 are highly similar. The jerangolid backbone is formed by a type I polyketide synthase (PKS), which incorporates 4 acetate and 4 propionate units (Figure 1).13 Remarkable deviations from the standard PKS processing are a tetrahydropyran (THP) cyclization in module 3, a C-methylation-olefin shift cascade in module 4, and a pyranone cyclization in module 7.14–16 The PKS product projerangolid (3) is proposed to undergo tailoring by the SAM-dependent O-methyltransferase JerF and the two Rieske FeS-cluster proteins JerP and JerL to finally give 1.13,17 The high density of steps catalyzed by non-canonical PKS domains and tailoring enzymes makes the jerangolid structure deviate substantially from that of common PKS I products, motivating in depth-studies on the pathway enzymology. Due to the characteristics of their biosynthesis, the jerangolids are predestines for a chemoenzymatic approach that exploits the aforementioned pathway enzymes to gain efficient access to derivative libraries.

Figure 1. a) Structures of jerangolid A (1) and ambruticin VS3 (2). b) Biosynthetic pathway of the jerangolids. The timing of the JerL/JerP-catalyzed oxidation steps is not elucidated. JerF: O-methyltransferase, JerO: monooxygenase for regeneration of JerL/JerP. Total syntheses of the pathway products jerangolid A (16 linear steps – 6% overall yield) and jerangolid D (12 linear steps – 14.5% overall yield) have been published.7,8 Both use olefination chemistry to couple preformed and protected building blocks at the double bonds of the skipped diene (Julia-Kocienski (JKO) olefination and phosphonamide-anion olefination). The pyranone was introduced as a stable methylenolether. For the DHP cyclization, the reactivity of the endocyclic double bond was exploited. Synthetic access to the remaining key intermediates projerangolid (3) and jerangolid E (4) has not yet been reported, though they are essential for the investigation of the tailoring steps. Both bear a THP instead of a DHP, necessitating another cyclization method. THPs are bulkier than the homologous DHPs, thus questioning the transferability of the previously established method for the formation of the (E)-configured double bond at C-9 and C-10. Additionally, projerangolid (3) exhibits a 3-methyl-6-vinyldihydro-2H-pyran-2,4(3H)-dione, which is considerably less stable than a cyclic methylenolether and cannot directly be coupled via JKO or related olefination reactions. A higher sensitivity of early tailoring intermediates compared to the end products of polyketide biosynthetic pathways is frequently

ACS Paragon Plus Environment

The Journal of Organic Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 13

observed and represents a general challenge for their synthesis.18– 20

Our strategy towards 3 and 4 envisaged closing the THP by an oxa-Michael addition to yield 8 after C1 degradation (Figure 2). This methylketone would be coupled with sulfone 7 in a JKO. Orthogonal olefin cross metathesis (OCM) was planned for joining vinylpyrone 6 and the JKO product. This rapid, protection groupfree endgame would occur under mild conditions, making it optimally suited for the synthesis of derivatives.

Figure 2. Retrosynthetic analysis of jerangolid E (4). Acetate aldol reaction followed by condensation with the freshly prepared potassium salt 12 yielded the 3-oxo-5-hydroxyester 10 (Scheme 1a).21,22 Lactone 11 was obtained by cyclization under weakly basic conditions and methylenolether 6 after additional Omethylation using Me2SO4. Homoallylic alcohol 14 is readily available in 3 steps from Oppolzer sultam 13 on a multigram scale (Scheme 1b).23,24 Sulfone 7 was synthesized from 14 via Mitsunobu reaction and oxidation of the thioether.25 For the synthesis of the pyranylester 18, we adapted a previously reported route from our group (Scheme 1c).14 Wittig olefination of aldehyde 16 with a stabilized phosphorane followed by TBS-deprotection afforded 7-hydroxy-2-enoate 17. Intramolecular oxa-Michael addition took place upon treatment with KOtBu resulting in formation of 18 as a mixture of the C-2 epimers. The lack of stereocontrol at this center was inconsequential as 18 was then converted into the methylketone 8 in four steps.26 . Due to the volatility and instability of the intermediates, the degradation sequence was carried out straightforwardly, thereby largely avoiding column chromatography.

Scheme 1. Synthesis of the building blocks 6, 11, 7 and 8.

As anticipated, the sterically demanding environment of the (E)configured double bond represented a significant challenge (Table 1). As OCM of olefin 19 with various terminal olefins was not successful, we turned our attention towards the optimization of JKO between 7 and 8. In a screening of various sulfones and bases, only phenylsulfone 7 produced olefination product in significant yields upon deprotonation with LiHMDS in CH2Cl2 (Table 1). A selectivity of up to 3.2:1 in favor of the (E)-configured product 20 was observed (entry 3). By adding CeCl3, a maximum yield of 73% and an E/Z ratio of 2.3:1 were achieved on a preparative useful scale (entry 5).27,28 Upscaling at acceptable losses in yield and stereoselectivity was possible to a scale of 100 mg (588 µmol) of starting material 8 (entries 6-9). The isomers were readily separable by flash chromatography and the unreacted ketone could be reisolated for further rounds of reaction. (Z)-Configured isomer 21 was used for the synthesis of a non-natural jerangolid E derivative (vide infra).

Table 1. Optimization of the JKO conditions. Relevant changes between the entries are highlighted in bold.a

entry

conditions

yield

E/Z

1

1.05 equiv. LiHMDS CH2Cl2, -78 °C to 0 °C, 5 h reaction scale: 20 mg

57%

2.1:1

2

1.3 equiv. LiHMDS CH2Cl2, -40 °C to rt, 17 h reaction scale: 17 mg

26%

2.6:1


Page 3 of 13 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


3

1.4 equiv. LiHMDS CH2Cl2, -40 °C to -20 °C, 3 h reaction scale: 10 mg

n/a

3.2:1

4

1.2 equiv. LiHMDS, 0.5 equiv. CeCl3 CH2Cl2, -40 °C to rt, 4 h (Barbier conditions) reaction scale: 10 mg

66%

1.5:1

5

1.6 equiv. LiHMDS, 0.4 equiv. CeCl3 CH2Cl2, -40 °C to rt, 6 h reaction scale: 18 mg

73%

2.3:1

6


54%

2.3:1

7


54%

2.0:1

8


61%

2.1:1

9


45%

1.8:1

a1.2

between pyrandione 11 and diene 20 gave only hardly reproducible results and low overall yields (Scheme 3). A considerably lower solubility of 11 compared to 6 as well as instability of 11 and 3 at elevated temperature were identified as mayor problems. Furthermore, the amphipathic nature of 3 and the sensitivity of the pyrandione moiety complicated purification by flash chromatography and semipreparative RP-HPLC. To circumvent these issues, we rearranged the endgame and coupled precursor 10 with 20 to obtain linear 24 in 86% yield. Exposure to mild cyclization conditions enabled isolation of projerangolid (3) in form of a mixture of its C-2 epimers in 79% yield after rapid purification by reverse phase flash chromatography. Using the same strategy, the derivative 5-epi-projerangolid (25) was produced starting from 20 and 5-epi-10 in two steps with a crude overall yield of 56%. After mildly acidic workup and extraction, the material was obtained in very good purity, which was sufficient for full characterization and bioconversion experiments.

Scheme 3. Synthesis of 3 and 25.

equiv. of 7 and 1.0 equiv. of 8 were generally applied.

Reacting an excess of the less reactive olefin 6, JKO product 20 and 2nd generation Grubbs catalyst led to the isolation of jerangolid E (7) in a yield of 23%.29,30 In contrast, using the Grela catalyst 23 in perfluorinated toluene delivered the trans-configured target compound 4 in 93% isolated yield.31,32 Analogously, diene 21 and lactone 6 were reacted with catalyst 23 to give the first described non-natural jerangolid derivative 9-(Z)-jerangolid E (22) in a yield of 76%.

Scheme 2. Synthesis of 4 and 22.

Synthetic projerangolid (3) and jerangolid E (4) were used to confirm the proposed role of JerF, for which we had previously shown activity on simple pyrandiones.17 A bioconversion experiment with 3 and heterologously expressed JerF showed complete conversion into 4 , supporting that JerF acts as the first tailoring enzyme of the jerangolid pathway (Figure 3, Figures S5-S18). 5-epi-Projerangolid (25) was also fully methylated by JerF, showing substrate tolerance for projerangolid-like derivatives with relevant structural changes near the methylation site (Figures S19-S24). Time course experiments showed that the individual reactions of both, 25 and 3, with JerF were essentially complete after 40 min and that the enzyme hardly differentiates between both compounds (Figure 3b/c, Figures S25-S41). The reaction was tolerant towards the addition of organic co-solvents. The conversion of 3 remained largely unaffected by DMSO proportions of up to 50% (Figure 3d, Figures S42S50). Taken together, substrate tolerance, high activity and robustness were observed in the JerF-conversions, designating the enzyme as a promising candidate for chemoenzymatic synthesis.

When we attempted to apply the established OCM conditions for the synthesis of projerangolid (3), several repetitions of the reaction



Page 4 of 13

Figure 3. a: UPLC-MS analysis of the bioconversion of 6 by JerF. b: Time course experiment of 3 and JerF. c: Comparison of the time course between 3 and 25. d: Tolerance of JerF towards varying proportions of DMSO. For conditions, see experimental section. In summary, we completed the total synthesis of the late-stage biosynthetic intermediates jerangolid E (4) (15 linear steps, 15.0 % overall yield) and projerangolid (3) (16 linear steps, 10.6% overall yield). Key steps were a JKO to join 7 and 8 as well as an OCM for the final fragment coupling. The established routes were used to obtain the non-natural derivatives 9-(Z)-jerangolid E (22) (3.2% overall yield) and 5-epi-projerangolid (25) (8.9% crude overall yield). Bioconversion experiments of synthetic 3 and 25 with JerF confirmed that this enzyme acts as the first tailoring enzyme of the jerangolid A pathway. Relaxed substrate specificity and tolerance to organic solvents was also observed, thus designating JerF as a potential tool for chemoenzymatic synthesis. Our results lay the foundation for a comprehensive investigation of all late-stage enzymes of the jerangolid biosynthetic pathway, which is an important milestone for the development of a chemoenzymatic system for derivative synthesis. Compounds 3, 4, 22 and 25 will undergo biological activity testing to assess the effect of structural variations, particularly within the pharmacophoric unit of the jerangolids. The flexible, modular nature of the synthetic route will allow the rapid synthesis of jerangolid derivatives in the future.

EXPERIMENTAL SECTION Chemistry Methods and Materials. Unless otherwise stated, all reactions were carried out in oven dried glassware under nitrogen or argon atmosphere using dry solvents and reagents. Solvents and reagents were purchased from Sigma-Aldrich, Acros Organics, ABCR or Roth. All NMR spectra were recorded with Bruker AVANCE DRX-500, DPX-400 or AVANCE-400 with the residual solvent signal as internal standard: CDCl3 7.26 ppm for 1H, 77.16 ppm for 13C; MeOD 3.31 ppm for 1H, 49.00 ppm for 13C; D2O 4.79 ppm for 1H. Signal multiplicities are stated, using the following abbrevations: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, b = broad signal. 13C NMR spectra are reported as values in ppm relative to residual solvent signal (abbrevations: q = quarternary carbon, t = tertiary carbon, s = secondary carbon, p = primary carbon). High resolution mass spectra (HRMS) were obtained with a Micromass LCT via loop-mode injection from a

Waters (Alliance 2695) HPLC system or a Thermo Fisher Scientific Q Exactive™ (Orbitrap) spectrometer with an UltiMate 3000TM UHPLC system. Alternatively, a Micromass Q-TOF in combination with a Waters Aquity Ultraperformance LC system was employed. Ionization was achieved by ESI. Modes of ionization, calculated and found mass are given. Reversed phase UPLCMS applications were performed with Waters Acquity Ultra Performance LC (column: ACQUITY UPLC® HSS C18 1.8 µm, 2.1 x 50 mm). Semi-preparative HPLC was perfomed with a Waters 600 HPLC system (Waters 2487 Dual λ Absorbance Detector). Solvents, operating procedures and retention times are given with the corresponding experimental and analytical data. Reversed phase HPLC and UPLC applications were performed with membrane-filtrated and double distilled water as well as commercial avaiblae HPLC grade solvents (all solvents were degassed by ultrasound). Optical rotations were measured with a polarimeter Typ 341 from Perkin-Elmer in a 10 cm quartz glass cuvette at λ = 589.3 mm (sodium-D-line). Notation rotations was done in 101∙cm2g-1 with concentration c in 10 mg∙mL-1 by definition. Reaction progress was followed by thin layer chromatography (MacheryNagel, ALUGRAM Xtra G/UV254) visualizing with UV light (254 nm) and KMnO4. Flash column chromatography was performed with silica gel (60 Å, 35-70 µm) from Merck. Solvents and retardation factor (Rf) are given with the corresponding experimental and analytical data (Abbrevations: PE = petroleum ether; Hex = hexane, EE = ethyl acetate). Biochemistry Methods and Materials. All chemicals and antibiotics were purchased from Sigma-Aldrich or Roth. For cultivation 2TY medium was used containing 1 % glucose and 135 µM ampicillin. Cell disruption was conducted by sonication (Sonoplus Typ HD 3100 from Bandelin) for 7 cycles (45% amplitude, 0.02 kJ/s, 30 s pulse and 30 s pause). Total protein concentration was measured via Bradford assay.


Page 5 of 13 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(R,E)-3-Hydroxy-1-((R)-4-isopropyl-2-thioxothiazolidin-3yl)hex-4-en-1-one (S11). TiCl4 (14.4 mL, 14.4 mmol, 1 equiv., 1 M in CH2Cl2) was added to a solution of (S)-Nagao-Auxilliary 9 (2.93 g, 14.4 mmol, 1 equiv.) in CH2Cl2 (150 mL) at -78 °C over 30 min. After 20 min DIPEA (3.01 mL, 17.3 mmol, 1.2 equiv.) was added dropwise over 20 min and stirring was continued for 1 h at -78 °C. Then crotonaldehyde (1 mL, 12.1 mmol, 0.9 equiv.) was added and stirring was continued 1 h. The reaction was quenched by addition of saturated NH4Cl-solution and stirring was continued for 1 h at room temperature. The aqueous layer was extracted three times with CH2Cl2, the combined organic layers were dried over MgSO4 and concentrated under reduced pressure. Flash chromatography (Hex/EE 8:1→4:1) of the crude product yielded aldol product S11 (2.50 g, 9.14 mmol, 75 %) as yellow oil. 1H NMR (500 MHz, CDCl3): δ 5.76 (dqd, J = 15.3, 6.5, 1.2 Hz, 1 H, 4-CH), 5.56 (ddq, J = 15.3, 6.5, 1.6 Hz, 1 H, 5-CH), 5.16 (ddd, J = 7.9, 6.3, 1.2 Hz, 1 H, 9-CH), 4.62 (m, 1 H, 3-CH), 3.62 (dd, J = 17.7, 2.9 Hz, 1 H,2-CH2), 3.52 (dd, J = 11.5, 8.0 Hz, 8-CH2), 3.30 (dd, J = 17.7, 9.0 Hz, 1 H, 2-CH2), 3.04 (dd, J = 11.6, 1.1 Hz, 1 H, 8CH2), 2.37 (dsept, J = 6.7, 6.8 Hz, 1 H, 10-CH), 1.71 (d, J = 6.4 Hz, 3 H, 6-CH3), 1.07 (d, J = 6.7 Hz, 3 H, 11‘-CH3), 0.99 (d, J = 7.0 Hz, 3 H, 11-CH3) ppm; 13C NMR (125 MHz, CDCl3): δ 203.1 (q, 7-C), 172.8 (q, 1-C), 131.8 (t, 4-CH), 127.6 (t, 5-CH), 71.5 (t, 9-CH), 68.9 (t, 3-CH), 45.6 (s, 2-CH2), 31.0 (s, 8-CH2), 30.8 (t, 10-CH), 19.2 (p, 6-CH3), 18.0 (p, 11-CH3), 17.9 (p, 11’-CH3) ppm; HRMS [ESI] m/z for C12H18NOS2 [M-H2O+H]+: calcd 256.0824, found 256.0827; [α]D20: +239.8 (c = 0.5, CH2Cl2); Rf (PE/EE 2:1): 0.70.

Ethyl-(5R,E)-5-hydroxy-2-methyl-3-oxooct-6-enoate (10). Potassium-3-ethoxy-2-methyl-3-oxopropanoate 12 (1.61 g, 8.74 mmol, 2.2 equiv.) and MgCl2 (415 mg, 4.37 mmol, 1.1 equiv.) were added to a solution of aldol product S11 (1.09 g, 3.97 mmol, 1 equiv.) in THF (10 mL) at room temperature. The resulting suspension was rapidly stirred for 45 min. Imidazole (297 mg, 4.37 mmol, 1.1 equiv.) was added and the stirring continued at room temperature. After 67 h the initial strongly yellow color faded. The reaction mixture was diluted with EtOAc, washed with 0.5 M HCl and the aqueous layer extracted three times with EtOAc. The combined organic layers were washed with 0.5 M NaHCO3 and the aqueous layer was three times reextracted with EtOAc. The combined organic layers were dried over NaSO4 and the solvent removed under reduced pressure. Flash chromatography (Hex/EE 6:1) yielded product 10 (850 mg, 3.97 mmol, quant.) as diastereomeric mixture in form of a colorless oil. 1H NMR (400 MHz, CDCl3): δ 5.73 (dqd, J = 15.4, 6.6, 0.9 Hz, 1 H, 7-CH), 5.48 (m 1 H, 6 H), 4.53 (m, 1 H, 5-CH), 4.19 (q, J = 7.1 Hz, 2 H, OEt), 3.53, 3.53 (q, J = 7.2 Hz, 1 H, 2-CH), 2.75 (m, 2 H, 4-CH2), 1.69 (d, J = 6.5 Hz, 3 H, 8-CH3), 1.35, 1.34 (d, J = 7.2 Hz, 3 H, 9CH3), 1.27 (t, J = 7.1 Hz, 3 H, OEt) ppm; 13C NMR (125 MHz, CDCl3): δ 206.3, 206.3 (q, 3-C), 170.4, 170.3 (q, 1-C), 131.9, 131.8 (t, 6-CH), 127.6, 127.5 (t, 7-CH), 68.7, 68.6 (t, 5-CH), 61.6, 61.6 (s, OEt), 53.6, 53.6 (t, 2-CH), 48.3, 48.1 (s, 4-CH2), 17.8 (p, 8CH3),14.2 (p, OEt), 12.7, 12.6 (p, 9-CH3) ppm; HRMS [ESI] m/z for C10H17O3 [M-H2O+H]+: calcd 197.1172, found 197.1171; Rf (PE/EE 4:1): 0.20.

(R,E)-4-Methoxy-3-methyl-6-(prop-1-en-1-yl)-5,6-dihydro2H-pyran-2-one (6). K2CO3 (812 mg, 5.88 mmol, 2 equiv.) was added to a solution of ketoester 10 (630 mg, 2.94 mmol, 1 equiv.) in MeOH (17 mL) at room temperature. After 1.5 h the solvent was removed under reduced pressure. The residue was taken up in acetone (17 mL), Me2SO4 (560µL, 5.88 mmol, 2 equiv.) was added and stirring was continued for 38 h at room temperature. The reaction mixture was diluted with EtOAc and washed with 0.1 M HCl. The aqueous layer was extracted three times with EtOAc, the combined organic layers were dried over NaSO4 and concentrated under reduced pressure. Flash chromatography (PE/EE 2:1) gave lactone 6 (501 mg, 2.75 mmol, 94 %). 1H NMR (500 MHz, CDCl3): δ 5.87 (dqd, J = 15.4, 6.5, 1.0 Hz, 1 H, 7-CH), 5.62 (ddq, J = 15.3, 7.0, 1.7 Hz, 1 H, 6-CH), 4.74 (m, 1 H, 5-CH), 3.78 (s, 3 H, OMe), 2.62 (ddq, J = 16.7, 4.3, 1.2 Hz, 1 H, 4-CH2), 2.55 (ddq, J = 16.9, 11.0, 1.9 Hz, 1 H, 4-CH2), 1.78 (dd, J = 1.9, 1.2 Hz, 3 H, 9-CH3), 1.74 (ddd, J = 6.6, 1.6, 0.7 Hz, 3 H, 8-CH3) ppm; 13C NMR (125 MHz, CDCl3): δ 168.4 (q, 3-C), 165.1 (q, 1-C), 130.8 (t, 7CH), 128.3 (t, 6-CH), 103.8 (q, 2-C), 75.2 (t, 5-CH), 55.6 (p, OMe), 29.8 (s, 4-CH2), 17.9 (p, 8-CH3), 9.0 (p, 9-CH3) ppm; HRMS [ESI] m/z for C10H15O3 [M+H]+: calcd 183.1016, found 183.1012; [α]D20: +50.4 (c = 1.0, CH2Cl2); Rf (PE/EE 2:1): 0.34.

(6R)-3-Methyl-6-((E)-prop-1-en-1-yl)dihydro-2H-pyran2,4(3H)-dione (11). K2CO3 (190 mg, 1.37 mmol, 2 equiv.) was added to a solution of ketoester 10 (147 mg, 0.69 mmol, 1 equiv.) in MeOH (4 mL) at room temperature. After 1.5 h the solvent was removed under reduced pressure. The residue was taken up in 1 M HCl, the aqueous layer extracted three times with EtOAc and the combined organic layers dried over NaSO4. After removal of the solvent under reduced pressure lactone 11 (112 mg, 0.67 mmol, 97 %) was obtained as a mixture of the C-2 epimers in sufficient purity as slightly yellowish solid. 1H NMR (500 MHz, CDCl3): δ 5.94 (dqd, J = 15.3, 6.6, 1.1 Hz, 1 H, 7-CH), 5.58 (ddq, J = 15.3, 6.9, 1.7 Hz, 1 H, 6-CH), 5.14 (m, 1 H, 5-CH), 3.58 (q, J = 6.6 Hz, 1 H, 2-CH), 2.77 (dd, J = 19.0, 3.0 Hz, 1 H, 4-CH2), 2.56 (dd, J = 19.0, 11.7 Hz, 1 H, 4-CH2), 1.79 (ddd, J = 6.6, 0.6, 1.7 Hz, 3 H, 8-CH3), 1.37 (d, J = 6.6 Hz, 3 H, 9-CH3) ppm; 13C NMR (125 MHz, CDCl3): δ 201.4 (q, 3-C), 169.7 (q, 1-C), 132.2 (t, 6CH), 126.5 (t, 7-CH), 74.8 (t, 5-CH), 51.9 (t, 2-CH), 43.5 (s, 4CH2), 17.9 (p, 8-CH3), 8.0 (p, p-CH3) ppm; MS [ESI] m/z for C9H13O3 [M+Na]+: calcd 169.0859, found 169.0855; [α]D20: +64.1 (c = 1.0, CH2Cl2); Rf (PE/EE 2:1): 0.32.

(S,E)-3-Hydroxy-1-((R)-4-isopropyl-2-thioxothiazolidin-3yl)hex-4-en-1-one (S15). TiCl4 (9.84 mL, 9.84 mmol, 1 equiv., 1 M in CH2Cl2) was added to a solution of (R)-Nagao-Auxilliary S14 (2.00 g, 9.84 mmol, 1 equiv.) in CH2Cl2 (100 mL) at 78 °C. After 10 min DIPEA (2.06 mL, 11.8 mmol, 1.2 equiv.) was added dropwise over 20 min and stirring was continued for 1 h at 78 °C. Then crotonaldehyde (731 µL, 8.86 mmol, 0.9 equiv.) was added and stirring was continued 1 h. The reaction was quenched



by addition of saturated NH4Cl-solution and stirring was continued for 1 h at room temperature. The aqueous layer was extracted three times with CH2Cl2, the combined organic layers were dried over MgSO4 and concentrated under reduced pressure. Flash chromatography (Hex/Et2O 7:1→4:1) of the crude product yielded aldol product S15 (2.08 g, 7.61 mmol, 77 %) as yellow oil. 1H NMR (500 MHz, CDCl3): δ 5.76 (dqd, J = 15.3, 6.5, 1.2 Hz, 1 H, 4-CH), 5.56 (ddq, J = 15.3, 6.5, 1.6 Hz, 1 H, 5-CH), 5.16 (ddd, J = 7.9, 6.3, 1.2 Hz, 1 H, 9-CH), 4.62 (m, 1 H, 3-CH), 3.62 (dd, J = 17.7, 2.9 Hz, 1 H,2-CH2), 3.52 (dd, J = 11.5, 8.0 Hz, 8-CH2), 3.30 (dd, J = 17.7, 9.0 Hz, 1 H, 2-CH2), 3.04 (dd, J = 11.6, 1.1 Hz, 1 H, 8CH2), 2.37 (dsept, J = 6.7, 6.8 Hz, 1 H, 10-CH), 1.71 (d, J = 6.4 Hz, 3 H, 6-CH3), 1.07 (d, J = 6.7 Hz, 3 H, 11‘-CH3), 0.99 (d, J = 7.0 Hz, 3 H, 11-CH3) ppm; 13C NMR (125 MHz, CDCl3): δ 203.1 (q, 7-C), 172.8 (q, 1-C), 131.8 (t, 4-CH), 127.6 (t, 5-CH), 71.5 (t, 9-CH), 68.9 (t, 3-CH), 45.6 (s, 2-CH2), 31.0 (s, 8-CH2), 30.8 (t, 10-CH), 19.2 (p, 6-CH3), 18.0 (p, 11-CH3), 17.9 (p, 11’-CH3) ppm; HRMS [ESI] m/z für C12H20NO2S2 [M+H]+: calcd 274.0930, found 274.0925; [α]D20: -293.5 (c = 0.4, CH2Cl2); Rf (PE/EE 8:1): 0.07.

Ethyl-(5S,E)-5-hydroxy-2-methyl-3-oxooct-6-enoate (5-epi10). Potassium-3-ethoxy-2-methyl-3-oxopropanoate 12 (555 g, 3.01 mmol, 2.0 equiv.) and MgCl2 (143 mg, 1.51 mmol, 1.0 equiv.) were added to a solution of aldol product S15 (411 mg, 1.51 mmol, 1 equiv.) in THF (3.76 mL) at room temperature. The resulting suspension was rapidly stirred for 30 min. Imidazole (103 mg, 1.51 mmol, 1.0 equiv.) was added and the stirring continued at room temperature. After 20 h the initial strongly yellow color faded. The reaction mixture was diluted with EtOAc, washed with 0.5 M HCl and the aqueous layer extracted three times with EtOAc. The combined organic layers were washed with 0.5 M NaHCO3 and the aqueous layer was three times reextracted with EtOAc. The combined organic layers were dried over NaSO4 and the solvent removed under reduced pressure. Flash chromatography (PE/EE 5:1) yielded product 5-epi-10 (166 mg, 0.77 mmol, 52 %) as a mixture of the C-2 epimers in form of a colorless oil. 1H NMR (400 MHz, CDCl3): δ 5.73 (dqd, J = 15.4, 6.6, 0.9 Hz, 1 H, 7-CH), 5.48 (m 1 H, 6 H), 4.53 (m, 1 H, 5-CH), 4.19 (q, J = 7.1 Hz, 2 H, OEt), 3.53, 3.53 (q, J = 7.2 Hz, 1 H, 2-CH), 2.75 (m, 2 H, 4-CH2), 1.69 (d, J = 6.5 Hz, 3 H, 8-CH3), 1.35, 1.34 (d, J = 7.2 Hz, 3 H, 9CH3), 1.27 (t, J = 7.1 Hz, 3 H, OEt) ppm; Rf (PE/EE 4:1): 0.14.

(E)-1-((3aR,6R)-8,8-Dimethyl-2,2-dioxidotetrahydro-3H-3a,6methanobenzo[c]isothiazol-1(4H)-yl)but-2-en-1-one (S17). Crotonic acid (30.0 g, 348 mmol, 3.0 equiv.) and DMAP (7.10 g, 58.0 mmol, 0.5 equiv.) were added to a solution of sultam 13 (25.0 g, 116 mmol, 1.0 equiv.) in CH2Cl2 (500 mL). DCC (26.4 g, 128 mmol, 1.1 equiv.) was added slowly at 0 °C. The reaction was stirred for 3 h at 0 °C before it was warmed to room temperature and stirred for 3 days. Concentration under reduced pressure gave a yellowish crude product. Two times recrystalization from MeOH gave product S17 (16.0 g, 56.0 mmol, 48 %) in sufficient purity. 1H NMR (500 MHz, CDCl3): δ 7.10 (dq, J = 15.0, 7.0 Hz, 1 H, 12CH), 6.59 (dq, J = 15.0, 1.7 Hz, 1 H, 11-CH), 3.93 (dd, J = 7.6, 5.0 Hz, 1 H, 7-CH), 3.48 (m, 2 H, 1-CH2), 2.17-2.06 (m, 2 H, 6CH2), 1.96-1.85 (m, 3 H, 3-CH2, 4-CH2, 5-CH), 1.93 (dd, J = 7.0,

Page 6 of 13

1.7 Hz, 3 H, 13-CH3), 1.45-1.32 (m, 2 H, 3-CH2, 4-CH2), 1.17 (s, 3 H, 9-CH3), 0.97 (s, 3 H, 9’-CH3) ppm; 13C NMR (125 MHz, CDCl3): δ 164.1 (q, 10-C), 146.3 (t, 12-CH), 122.4 (t, 11-CH), 65.3 (t, 7-CH), 53.3 (s, 1-CH2), 48.6 (q, 2-C), 47.9 (q, 8-C), 44.8 (t, 5CH), 38.6 (s, 6-CH2), 33.0 (s, 3-CH2), 26.6 (s, 4-CH2), 21.0 (p, 9CH3), 20.0 (p, 9’-CH3), 18.5 (p, 13-CH3) ppm; HRMS [ESI] m/z für C14H21NO3SNa [M+Na]+: calcd 306.1134, found 306.1132; [α]D20: -83.8 (c = 1.0, CH2Cl2); Rf (Hex/EE 7:3): 0.74. The analytical data are in agreement with those in the literature.33

(2R)-1-((3aR,6R)-8,8-Dimethyl-2,2-dioxidotetrahydro-3H3a,6-methanobenzo[c]isothiazol-1(4H)-yl)-2-methylbut-3-en-1one (S18). A solution of N-crotylbornan-10,2-sultam S17 (16.0 g, 56.5 mmol, 1 equiv.) in THF (450 mL) was added slowly over 2 h to a solution of LiHMDS (130 mL, 130 mmol, 2.3 equiv., 1 M in THF) and HMPA (68.5 mL, 390 mmol, 6.9 equiv.) at -78 °C. After 45 min a solution of MeI (24.3 mL, 390 mmol, 6.9 equiv.) in THF (120 mL) was added dropwise over 40 min. The reaction was quenched after 45 min by addition of saturated NH4Cl-solution (400 mL) and allowed to warm to room temperature. The aqueous layer was extracted three times with EtOAc. The combined organic layers were washed three times respectively with H2O and brine, dried over MgSO4 and concentrated under reduced pressure. Recrystallization from MeOH gave product S18 (14.6 g, 49.1 mmol, 87 %) as long clear needles. 1H NMR (500 MHz, CDCl3): δ 5.97 (ddd, J = 17.4, 10.2, 7.3 Hz, 1 H, 12-CH), 5.20 (ddd, J = 17.2, 1.2, 1.2 Hz, 1 H, 13-CH2), 5.14 (ddd, J = 10.3, 1.1, 1.1 Hz, 1 H, 13CH2), 3.88 (t, J = 6.4 Hz, 1 H, 7-CH), 3.79 (m, 1 H, 11-CH), 3.48 (m, 2 H, 1-CH2), 2.06 (m, 2 H, 6-CH2), 1.95-1.84 (m, 3 H, 3-CH2, 4-CH2, 5-CH), 1.43-1.35 (m, 2 H, 3-CH2, 4-CH2), 1.34 (d, J = 6.9 Hz, 3 H, 14-CH3), 1.16 (s, 3 H, 9‘-CH3), 0.97 (s, 3 H, 9CH3) ppm; 13C NMR (125 MHz, CDCl3): δ 174.4 (q, 10-C), 136.4 (t, 12-CH), 116.7 (s, 13-CH2), 65.3 (t, 7-CH), 53.3 (s, 1-CH2), 48.5 (q, 2-C), 47.9 (q, 8-C), 44.7 (t, 5-CH), 44.1 (t, 11-CH), 38.5 (s, 6-CH2), 33.0 (s, 3-CH2), 26.6 (s, 4-CH2), 21.0 (p, 9‘-CH3), 20.0 (p, 9-CH3), 19.0 (p, 14-CH3) ppm; HRMS [ESI] m/z for C15H24NO3S [M+H]+: calcd 298.1471, found 298.1467; [α]D20: -75.9 (c = 1.0, CH2Cl2). The analytical data are in agreement with those of the enantiomer in the literature except for the optical rotation.34

(R)-2-Methylbut-3-en-1-ol (14). A solution of sultam S18 (15.2 g, 51.1 mmol, 1 equiv.) in Et2O (450 mL) was added dropwise to a supension of LiAlH4 (4.24 g, 106 mmol, 2.1 equiv.) in Et2O (69 mL) at 0 °C over 4.5 h. After complete addition the reaction mixture was warmed to room temperature and stirred for 1 h. The reaction was quenched by careful subsequent addition of H2O (4.3 mL), 15 % NaOH (4.3 mL) and H2O (13 mL). The mixture was dried over NaSO4 for 12 h and filtered through a glass frit. The filter cake was suspended in Et2O (170 mL), filtered again and filtrates combined. This procedure was repeated four times. Et2O was removed via atmospheric distillation using a vigreux column. Vacuum distillation of the residue gave alcohol 14 (3.20 g, 37.2 mmol, 73 %). 1H NMR (500 MHz, CDCl3): δ 5.71 (ddd, J = 17.5, 10.2, 7.3 Hz, 1 H, 3-CH), 5.13 (m, 1 H, 4-CH2), 5.10 (m, 1 H, 4-CH2), 3.52 (dd, J = 10.6, 5.5 Hz, 1 H, 1-CH2), 3.43 (dd, J = 10.5, 7.6 Hz, 1 H, 1-CH2), 2.37 (dtq, J = 6.9, 6.9, 6.8 Hz, 1 H, 2-CH), 1.02 (d, J = 6.7 Hz, 3 H, 5-CH3) ppm; 13C NMR (125 MHz, CDCl3): δ 141.0 (t, 3-CH), 116.0 (s, 4-CH2), 67.1 (s, 1-CH2), 40.8 (t, 2-CH),


Page 7 of 13 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


16.1 (p, 5-CH3) ppm; [α]D20: +33.1 (c = 1.0, CH2Cl2); b.p.: 58 °C (95 mbar). The analytical data are in agreement with those of the enantiomer in the literature.34

(R)-5-((2-Methylbut-3-en-1-yl)thio)-1-phenyl-1-H-tetrazole (S19). PPh3 (3.05 g, 11.6 mmol, 1.2 equiv.) and 1-phenyl1H –tetrazole-5-thiol (2.07 g, 11.6 mmol, 1.2 equiv.) were added to a solution of alcohol 14 (1 mL, 9.70 mmol, 1 equiv.) in THF (96 mL) at 0 °C. DIAD (2.28 mL, 11.6 mmol, 1.2 equiv.) was added dropwise and the reaction was slowly warmed to room temperature. After 1.5 h the reaction was quenched by addition of saturated NH4Cl-solution. The aqueous layer was extracted three times with Et2O, the combined organic layers were dried over MgSO4 and concentrated under reduced pressure. Filtration through a pad of silica (PE/EE 10:1) the colorless oil S19 (2.79 g) was obtained in sufficient purity for the next step. 1H NMR (500 MHz, CDCl3): δ 7.60-7.52 (m, 5 H, Ph), 5.76 (ddd, J = 17.4, 10.3, 7.2 Hz, 1 H, 2-CH), 5.09 (ddd, J = 17.2, 1.2, 1.3 Hz, 1 H, 1CH2), 5.05 (ddd, J = 10.3, 1.2, 1.2 Hz, 1 H, 1-CH2), 3.42 (m, 2 H, 4-CH2), 2.68 (dtq, J = 6.8, 7.0, 6.9 Hz, 1 H, 3-CH), 1.18 (d, J = 6.8 Hz, 3 H, 5-CH3) ppm; 13C NMR (125 MHz, CDCl3): δ 154.7 (q, 6-C), 141.0 (t, 2-C), 133.9 (q, Ph), 130.2 (t, Ph), 129.9 (t, Ph), 124.0 (t, Ph), 115.5 (s, 1-C), 39.6 (s, 4-C), 37.5 (t, 3-C), 19.5 (p, 5-C) ppm; HRMS [ESI] m/z for C12H15N4S [M+H]+: calcd 247.1012, found 247.1007; [α]D20: +16.0 (c = 0.5, CH2Cl2); Rf (PE/EE 8:1): 0.50. The analytical data are in agreement with those of the enantiomer in the literature except for the optical rotation.25

(R)-5-((2-Methylbut-3-en-1-yl)sulfonyl)-1-phenyl-1-H-tetrazole (7). To a solution of the crude product S19 from the previous step in EtOH (62 mL) a solution of ammonium molybdate (3.59 g, 2.91 mmol, 0.3 equiv.) in H2O2 (6 mL, 194 mmol, 20 equiv., 30%) was added dropwise at 0 °C. The reaction was closely followed via TLC. To prevent the formation of the epoxide the reaction was quenched early by addition of H2O after 19 h. The aqueous layer was extracted three times with CH2Cl2, the combined organic layers were dried over MgSO4 and concentrated under reduced pressure. Flash chromatography (PE/EE 6:1) gave sulfone 7 (1.60 g, 5.75 mmol, 60 %). In a second oxidation cycle remaining sulfoxide was converted to sulfone 7 (combined yield: 2.37 g, 8.52 mmol, 88 %). The initially colorless, highly viscous oil crystalized upon storage at -20 °C after several days. 1H NMR (500 MHz, CDCl3): δ 7.67-7.58 (m, 5 H, Ph), 5.74 (ddd, J = 17.3, 10.1, 7.4 Hz, 1 H, 2-CH), 5.10 (ddd, J = 17.0, 1.2, 1.2 Hz, 1 H, 1CH2), 5.03 (ddd, J = 10.2, 1.1 1.0 Hz, 1 H, 1-CH2), 3.91 (dd, J = 14.7, 7.2 Hz, 1 H, 4-CH2), 3.69 (dd, J = 14.5, 6.4 Hz, 1 H, 4CH2), 3.03 (dtq, J = 6.9, 7.0, 7.0 Hz, 1 H, 3-CH), 1.25 (d, J = 6.9 Hz, 3 H, 5-CH3) ppm; 13C NMR (125 MHz, CDCl3): δ 154.0 (q, 6-C), 139.6 (t, 2-CH), 133.2 (q, Ph), 131.6 (t, Ph), 129.8 (t, Ph), 125.3(t, Ph) 115.9 (s, 1-CH2), 61.4 (s, 4-CH2), 32.9 (t, 3CH), 20.1 (p, 5-CH3) ppm; HRMS [ESI] m/z for C12H15N4O2S [M+H]+: calcd 279.0910, found 279.0906; [α]D20: +21.6 (c = 0.4, CH2Cl2); Rf (PE/EE 6:1): 0.53. The analytical data are in agreement with those of the enantiomer in the literature except for the optical rotation.25

(6S,7R,E)-Ethyl-7-((tert-butyldimethylsilyl)oxy)-2,6-dimethylnon-2-enoate (S7). (Carbethoxyethylidene)triphenylphosphorane (5.37 g, 14.8 mmol, 2 equiv.) was added to a solution of aldehyde 16 (1.91 g, 7.41 mmol, 1 equiv.) in CH2Cl2 (35 mL). The reaction mixture was stirred for 22 h at room temperature before the solvent was removed under reduced pressure. Flash chromatography (PE/EE 50:1) of the crude product yielded ethylester S7 (2.19 g, 6.39 mmol, 86 %). 1H NMR (400 MHz, CDCl3): δ 6.75 (dt, J = 7.3, 1.4 Hz, 1 H, 3-CH), 4.18 (q, J = 7.2 Hz, 2 H, OEt), 3.43 (q, J = 5.5 Hz, 1 H, 7-CH), 2.28-2.18 (m, 1 H, 4-CH2), 2.16-2.04 (m, 1 H, 4-CH2), 1.83 (s, 3 H, 10-CH3), 1.63-1.56 (m, 1 H, 5-CH2), 1.53-1.46 (m, 1 H, 5-CH2), 1.44-1.36 (m, 2 H, 8-CH2), 1.29 (t, J = 7.2 Hz, 3 H, OEt), 1.26-1.19 (m, 1 H, 6-CH), 0.89-0.84 (m, 15 H, OTBS, 11-CH3, 9-CH3), 0.03 (s, 3 H, OTBS), 0.02 (s, 3 H, OTBS) ppm; 13C NMR (100 MHz, CDCl3): δ 168.3 (q, 1-C), 142.5 (t, 3-CH), 127.7 (q, 2-C), 77.2 (t, 7-CH), 60.4 (s, OEt), 37.5 (t, 6CH), 31.1 (s, 5-CH2), 26.7 (s, 4-CH2), 25.9 (p, OTBS), 25.2 (s, 8CH2), 18.2 (q, OTBS), 14.8 (p, 11-CH3), 14.3 (p, OEt), 12.3 (p, 10CH3), 10.2 (p, 9-CH3), -4.4 (p, OTBS), -4.5 (p, OTBS) ppm; HRMS [ESI] m/z for C19H38O3SiNa [M+Na]+: calcd 365.2488, found 365.2484. [α]D20: -2.2 (c = 1.0, CH2Cl2); Rf (PE/Et2O 5:1): 0.78.

(6S,7R,E)-Ethyl-7-hydroxy-2,6-dimethylnon-2-enoate (17). PPTS (14.2 g, 56.5 mmol, 4.2 equiv.) was added to a solution of ester S7 (4.66 g, 13.6 mmol, 1.0 equiv.) in MeOH (300 mL). After stirring for three days at 50 °C the solvent was removed under reduced pressure and the aquoues layer extracted three times with CH2Cl2. The combined organic layers were washed with saturated NaHCO3-solution and brine, dried over MgSO4 and the solvent was removed under reduced pressure. Flash chromatography (PE/EE 6:1) of the crude product yielded compound 17 (3.07 g, 13.5 mmol, 99 %). 1H NMR (200 MHz, CDCl3): δ 6.74 (m, 1 H, 3CH), 4.18 (q, J = 7.1 Hz, 2 H, OEt), 3.36 (m, 1 H, 7-CH), 2.19 (m, 2 H, 4-CH2), 1.83 (m, 3 H, 10-CH3), 1.72-1.37 (m, 4 H, 5-CH2, 8CH2), 1.35-1.20 (m, 1 H, 6-CH), 1.29 (t, J = 7.1 Hz, 3 H, OEt), 0.96 (t, J = 7.8 Hz, 3 H, 9-CH3), 0.92 (d, J = 6.8 Hz, 3 H, 11-CH3) ppm; 13C NMR (100 MHz, CDCl3): δ 168.4 (q, 1-C), 142.4 (t, 3CH), 127.9 (q, 4-CH2), 77.5 (t, 7-CH), 60.6 (s, OEt), 38.3 (t, 6-CH), 30.8 (s, 5-CH2), 26.5 (s, 8-CH2), 26.5 (s, 4-CH2), 15.4 (p, 11-CH3), 14.4 (p, OEt), 12.5 (p, 10-CH3), 10.5 (p, 9-CH3) ppm; HRMS [ESI] m/z for C13H24O3Na [M+Na]+: calcd 251.1623, found 251.1623; [α]D20: -6.3 (c = 1.2, CH2Cl2); Rf (PE/EE 5:1): 0.28.

Ethyl-2-((2R,5S,6R)-6-ethyl-5-methyltetrahydro-2H-pyranyl)-propanoate (18). KOtBu (269 mg, 2.40 mmol, 1 equiv.) was added to a solution of ester 17 (548 mg, 2.40 mmol, 1 equiv.) in CH2Cl2 (32 mL) at 0 °C. After stirring for 4 h the reaction was quenched by addition of saturated NH4Cl-solution. The aqueous layer was extracted three times with CH2Cl2, the combined organic layers were washed with brine and dried over MgSO4. Removal of the solvent under reduced pressure and subsequent flash chromatography (PE/EE 39:1) yielded pyrane 18 (488 mg, 2.14 mmol, 89 %) as diastereomeric mixture. 1H NMR (400 MHz, CDCl3): δ



4.13 (m, 2 H, OEt), 3.40 (m, 1 H, 3-CH), 2.83-2.76 (m, 1 H, 7-CH), 2.51-2.42 (m, 1 H, 2-CH), 1.82-1.61 (m, 2 H, 5-CH2, 6-CH2), 1.55 (m, 1 H, 4-CH2), 1.38-1.14 (m, 9 H, 4-CH2, 6-CH, 8-CH2, 10-CH3, 5-CH2, OEt), 1.09 (d, J = 7.2 Hz, 1 H, 10-CH3), 0.92, 0.87 (t, J = 7.3 Hz, 3 H, 9-CH3), 0.80, 0.79(d, J = 6.5 Hz, 3 H, 11-CH3) ppm; 13C NMR (100 MHz, CDCl3): δ 175.6, 175.0 (q, 1-C), 85.0, 84.8 (t, 7-CH), 79.5, 78.5 (t, 3-CH), 60.2 (s, OEt), 45.9, 45.8 (t, 2CH), 35.1, 35.0 (t, 6-CH), 32.8 (s, 5-CH2), 30.0, 28.9 (s, 4-CH2), 26.1, 25.9 (s, 8-CH2), 17.7, 17.6 (p, 11-CH3), 14.3, 14.2 (p, OEt), 13.4, 13.2 (p, 10-CH3), 9.9, 9.8 (p, 9-CH3) ppm; HRMS [ESI] m/z for C13H24O3Na [M+Na]+: calcd 251.1623, found 251.1623; Rf (PE/EE 39:1): diastereomer a: 0.07, b: 0.13.

2-((2R,5S,6R)-6-Ethyl-5-methyltetrahydro-2H-pyran-2-yl)propanol (S8). DIBAL-H (8.04 mL, 8.04 mmol, 4 equiv., 1 M in hexane) was cooled to 4 °C and then added slowly dropwise to a solution of ester 18 (459 mg, 2.01 mmol, 1.0 equiv.) in CH2Cl2 (35 mL) at -78 °C. After complete addition the reaction was stirred at 0 °C for 5 h before saturated K-Na-tartrate-solution (50 mL) was added and stirring continued at room temperature for 16 h. The aqueous layer was extracted three times with CH2Cl2, the combined organic layers were dried over MgSO4 and the solvent removed under reduced pressure. Flash chromatography (PE/EE 5:1) of the crude product yielded alcohol S8 (375 mg, 2.01 mmol, 99 %). 1H NMR (400 MHz, CDCl3): δ 3.71-3.59 (m, 2 H, 1-CH2), 3.51-3.45, 3.20 (m, 1 H, 3-CH), 2.92-2.84 (m, 1 H, 7-CH), 1.94 (m, 1 H, 2CH), 1.83-1.64 (m, 4 H, 2’-CH, 8-CH2, 4-CH2, 5-CH2), 1.53-1.45 (m , 1 H, 8-CH2), 1.43-1.29 (m, 3 H, 4-CH2, 4’-CH2, 6-CH), 1.221.11 (m, 1 H, 5-CH2), 0.97-0.89 (m, 4 H, 10-CH3, 9-CH3), 0.85 (m, 2 H, 10-CH3), 0.81 (m, 3 H, 11-CH3) ppm; 13C NMR (100 MHz, CDCl3): δ 85.1, 85.0 (t, 7-CH), 84.5, 81.9 (t, 3-CH), 68.5, 66.9 (t, 2-CH), 40.2, 38.7 (s, 1-CH2), 34.6 (t, 6-CH), 32.8, 32.7 (s, 5-CH2), 30.5, 27.0 (s, 4-CH2), 25.8 (s, 8-CH2), 17.7 (p, 11-CH3), 13.7, 11.8 (p, 10-CH3), 9.8, 9.6 (p, 9-CH3) ppm; HRMS [ESI] m/z for C11H22O2Na [M+Na]+: calcd 209.1517, found 209.1517; Rf (PE/EE 5:1): diastereomer a: 0.42, b: 0.53.

2-((2R,5S,6R)-6-Ethyl-5-methyltetrahydro-2H-pyran-2-yl)propylmethansulfonate (S9). DIPEA (1.86 mL, 10.7 mmol, 1.5 equiv.) and MsCl (824 µL, 10.7 mmol, 1.5 equiv.) were added to a solution of alcohol S8 (1.33 g, 7.10 mmol, 1.0 equiv.) in CH2Cl2 (45 mL) at 0 °C. The reaction mixture was allowed to warm to room temperature and stirred for 18 h. The organic layer was washed twice with H2O, dried over MgSO4 and concentrated under reduced pressure. Filtration through a pad of silica (PE/EE 10:1) gave a yellow oil (1.97 g) as crude product which was directly used for the next step. 1H NMR (500 MHz, CDCl3): δ 4.35-4.25, 4.10 (m, 2 H, 1-CH2) , 3.31, 3.10 (m, 1 H, 3-CH), 2.99, 2.99 (s, 3 H, OMs), 2.83-2.76 (m, 1 H, 7-CH), 1.95, 1.89 (m, 1 H, 2-CH), 1.79, 1.73-1.62 (m, 3 H, 4’-CH2, 5-CH2, 8-CH2), 1.50-1.10 (m, 4 H, 4CH2, 5-CH2, 6-CH, 8-CH2), 1.00 (m, 3 H, 9-CH3), 0.96-0.89 (m, 3 H, 10-CH3), 0.80 (m, 3 H, 11-CH3) ppm; HRMS [ESI] m/z for C12H24O4SNa [M+Na]+: calcd 287.1293, found 287.1293; Rf (PE/EE 10:1): 0.1.

Page 8 of 13

(2R,3S,6R)-2-Ethyl-3-methyl-6-(prop-1-en-2-yl)tetrahydro-2Hpyran (19). A solution of the crude product from the previous step S9 in DMF (190 mL) was protected against light and NaI (3.19 g, 21.3 mmol, 3 equiv.) was added in the dark. The reaction mixture was covered with additional alufoil and heated to 53 °C. After 18 h DBU (1.89 mL, 12.8 mmol, 1.8 equiv.) was added slowly and the temperature was increased to 83 °C. After 6 h the reaction mixture was poured into a seperatory funnel filled with crushed ice. The aqueous layer was extracted three times with pentane. The combined organic layers were washed subsequently with 1 M HCl and saturated NaHCO3-solution, dried over MgSO4 and concentrated under reduced pressure. Alkene 19 (1.3 g) was obtained as yellow oil in sufficient purity to be used without further purification. Alkene 19 shows a characteristic smell of burnt pinetree needles. 1H NMR (400 MHz, CDCl3): δ 4.97 (m, 1 H, 1-CH2), 4.80 (m, 1 H, 1-CH2), 3.65 (bd, J = 10.9 Hz, 1 H, 3-CH), 2.91 (ddd, J = 9.3, 7.9, 2.8 Hz, 1 H, 7-CH), 1.81 (m, 1 H, 4-CH2), 1.76 (s, 3 H, 10-CH3), 1.75-1.66 (m, 2 H, 5-CH2, 8-CH2), 1.47-1.19 (m, 4 H, 4CH2, 5-CH2, 6-CH, 8-CH2), 0.96 (t, J = 7.3 Hz, 3 H, 9-CH3), 0.82 (d, J = 6.6 Hz, 3 H, 11-CH3) ppm; 13C NMR (100 MHz, CDCl3): δ 146.8 (q, 2-C), 109.8 (s, 1-CH2), 84.6 (t, 7-CH), 80.4 (t, 3-CH), 34.6 (t, 6-CH), 33.3 (s, 5-CH2), 30.9 (s, 4-CH2), 26.1 (s, 8-CH2), 19.5 (p, 10-CH3), 17.9 (p, 11-CH3), 9.7 (p, 9-CH3) ppm; [α]D20: +1.4 (c = 0.7, CH2Cl2); Rf (PE/EE 50:1): 0.31.

1-((2R,5S,6R)-6-Ethyl-5-methyltetrahydro-2H-pyran-2yl)ethan-1-one (8). A continuous stream of Ozone was bubbled through a solution of alkene 19 from the previous step in CH2Cl2 (10 mL) at -78 °C until a consistent blue coloration was observed. Afterwards oxgen was bubbled through the solution until complete decoloration. was achieved. Me2S (547 µL, 7.46 mmol, 1.05 equiv.) was added and the reaction was allowed to warm to room temperature overnight. After 18 h the solvent was removed under reduced pressure and flash chromatography (PE/EE 15:1) of the crude product yielded ketone 8 (746 mg, 4.38 mmol, 62 % over three steps). 1H NMR (500 MHz, CDCl3): δ 3.71 (dd, J = 11.7, 2.2 Hz, 1 H, 3-CH), 2.90 (ddd, J = 9.2, 8.8, 2.5 Hz, 1 H, 7-CH), 2.21 (s, 3 H, 1-CH3), 1.85 (m, 2 H, 4-CH2, 5-CH2), 1.74 (dqd, J = 14.5, 7.3, 2.8 Hz, 1 H, 8-CH2), 1.44 (m, 1 H, 8-CH2), 1.41-1.31 (m, 2 H, 4-CH2, 6-CH), 1.26-1.16 (m, 1 H, 5-CH2), 0.98 (t, J = 7.4 Hz, 3 H, 9-CH3), 0.83 (d, J = 6.6 Hz, 3 H, 10-CH3) ppm; 13C NMR (125 MHz, CDCl ): δ 210.6 (q, 2-C), 84.7 (t, 7-CH), 83.2 3 (t, 3-CH), 34.5 (t, 6-CH), 32.5 (s, 5-CH2), 28.5 (s, 4-CH2), 25.9 (p, 1-CH3), 25.9 (s, 8-CH2), 17.6 (p, 10-CH3), 9.7 (p, 9-CH3) ppm; MS [ESI] m/z for C10H19O2 [M+H]+: calcd 171.1380, found 171.1376; [α]D20: +1.3 (c = 0.7, CH2Cl2); Rf (PE/EE 12:1): 0.44.

(2R,3S)-2-Ethyl-3-methyl-6-((R,E)-4-methylhexa-2,5-dien-2yl)tetrahydro-2H-pyran (20) & (2R,3S)-2-Ethyl-3-methyl-6((R,Z)-4-methylhexa-2,5-dien-2-yl)tetrahydro-2H-pyran (21). LiHMDS (134 µL, 134 µmol, 1.3 equiv., 1 M in THF) was added to a solution of sulfone 7 (34.0 mg, 124 µmol, 1.2 equiv.) in CH2Cl2 at -40 °C. After 10 min CeCl3 (11.1 mg, 45 µmol, 0.4 equiv.) was added, followed by dropwise addition of a solution of ketone 8 (17.5 mg, 103 µmol, 1.0 equiv.) in CH2Cl2 (510 µL). The reaction mixture was slowly warmed to room temperature and stirred for 4 h. The reaction mixture was cooled down to -14 °C and additional LiHMDS (30.0 µL, 30.0 µmol, 0.3 equiv.) was added.


Page 9 of 13 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Stirring was continued for 1 h 45 min before the reaction was quenched by addition of saturated NH4Cl-solution. The aqueous layer was extracted three times with CH2Cl2, the combined organic layers dried over MgSO4 and concentrated carefully under reduced pressure (200 mbar). Flash chromatography (PE/EE 40:1) of the crude product gave a mixture of E/Z-isomers (16.6 mg, 75.0 µmol, 73 %, E:Z 2.3:1). Separation of the isomers was achieved by flash chromatography (eluent: pentane). (E)-Isomer 20: 1H NMR (500 MHz, CDCl3): δ 5.76 (ddd, J = 17.2, 10.3, 6.2 Hz, 1 H, 2CH), 5.24 (ddq, J = 9.0, 1.3, 1.2 Hz, 1 H, 4-CH), 4.95 (ddd, J = 17.3, 1.7, 1.7 Hz, 1 H, 1-CH2), 4.89 (ddd, J = 10.2, 1.7, 1.6 Hz, 1 H, 1-CH2), 3.61 (bd, J = 10.9 Hz, 1 H, 6-CH), 3.08 (m, 1 H, 3CH), 2.90 (ddd, J = 9.7, 7.2, 2.7 Hz, 1 H, 10-CH), 1.79 (dddd, J = 12.8, 3.5, 3.4, 3.5 Hz 1 H, 8-CH2), 1.70 (m, 1 H, 11-CH2), 1.65 (d, J = 1.4 Hz, 3 H, 14-CH3), 1.62 (m, 1 H, 7-CH2), 1.48-1.38 (m, 2 H, 7-CH2, 11-CH2), 1.38-1.30 (m, 1 H, 9-CH), 1.24-1.15 (m, 1 H, 8-CH2), 1.06 (d, J = 6.9 Hz, 3 H, 13-CH3), 0.95 (t, J = 7.4 Hz, 3 H, 12-CH3), 0.81 (d, J = 6.6 Hz, 3 H, 15-CH3) ppm; 13C NMR (125 MHz, CDCl3): δ 143.1 (t, 2-CH), 136.3 (q, 5-C), 128.2 (4CH), 112.1 (s, 1-CH2), 84.4 (t, 10-CH), 82.1 (t, 6-CH), 36.0 (t, 3CH),34.3 (t, 9-CH), 33.4 (s, 8-CH2), 31.0 (s, 7-CH2), 26.0 (s, 11CH2), 20.6 (p, 13-CH3), 17.9 (p, 15-CH3), 13.1 (p, 14-CH3), 9.6 (p, 12-CH3) ppm; [α]D20: +3.1 (c = 0.5, CH2Cl2); Anal. Calcd for C15H26O: C, 81.02; H, 11.79; N, 0. Found: C, 77.56; H, 11.37; N, 0.12.; Rf (PE): 0.09. (Z)-Isomer 21: 1H NMR (500 MHz, CDCl3): δ 5.79 (ddd, J = 17.2, 10.3, 5.9 Hz, 1 H, 2-CH), 5.00 (ddd, J = 17.2, 1.7, 1.7 Hz, 1 H, 1-CH2), 5.00 (m, 1 H, 4-CH), 4.91 (ddd, J = 10.4, 1.6, 1.6 Hz, 1 H, 1-CH2), 4.10 (dd, J = 11.4, 2.2 Hz, 1 H, 6-CH), 3.19 (m, 1 H, 3-CH), 2.90 (ddd, J = 9.6, 6.9, 2.9 Hz, 1 H, 10-CH), 1.79 (dddd, J = 12.9, 3.6, 3.6, 3.1 Hz, 1 H, 8-CH2), 1.71 (d, J = 1.5 Hz, 3 H, 14-CH3), 1.70-1.62 (m, 1 H, 11-CH2), 1.62-1.56 (m, 1 H, 7-CH2), 1.45-1.38 (m, 2 H, 7-CH2, 11-CH2), 1.38-1.31 (m, 1 H, 9-CH), 1.28-1.18 (m, 1 H, 8-CH2), 1.02 (d, J = 6.9 Hz, 3 H, 13-CH3), 0.91 (t, J = 7.4 Hz, 3 H, 12-CH3), 0.81 (d, J = 6.6 Hz, 3 H, 15-CH3) ppm; 13C NMR (125 MHz, CDCl3): δ 143.3 (t, 2CH), 136.3 (q, 5-C), 130.1 (4-CH), 112.3 (s, 1-CH2), 84.1 (t, 10CH), 76.8 (t, 6-CH), 35..6 (t, 3-CH), 33.8 (t, 9-CH), 33.3 (s, 8-CH2), 30.8 (s, 7-CH2), 25.9 (s, 11-CH2), 20.9 (p, 13-CH3), 19.4 (p, 14CH3), 18.0 (p, 15-CH3), 9.3 (12-CH3) ppm; Anal. Calcd for C15H26O: C, 81.02; H, 11.79; N, 0. Found: C, 79.02; H, 11.69; N, 0.27.; [α]D20: +4.7 (c = 0.5, CH2Cl2); Rf (PE): 0.11.

Jerangolid E (4). A solution of lactone 6 (36.0 mg, 198 µmol, 3.38 equiv.) in C7F8 (156 µL) was added to alkene 20 (13.0 mg, 58.5 µmol, 1.00 equiv.). The solvent was degassed by bubbling argon through it for 5 min. Grela catalyst (2.4 mg, 3.6 µmol, 0.06 equiv.) was added, the reaction flask sealed with a glass stopper and heated to 88 °C. After 2 h the reaction was cooled down to room temperature, a second portion of Grela catalyst (2.4 mg, 3.6 µmol, 0.06 equiv.) added and the stirring continued for 2 h at 88 °C. The reaction mixture was cooled down to room temperature and flash chromatography (PE/EE 5:1) of the mixture yielded jerangolid E (4) (20.7 mg, 57.1 µmol, 98 %). The pure product was obtained by semipreparative RP-HPLC purification in several batches (average recovery: 95%, isolated yield: 93%). RP-HPLC purification: column: Kinetex C18 5 µm, 100 Å; gradient (H2O/MeOH): 95/5-25/75-5/95, 5 min-20 min-26 min, flow: 20 ml/min; HRMS [ESI] m/z for C22H34O4Na [M+Na]+: calcd 385.2349, found 385.2342, C22H35O4 [M+H]+: 20 calcd 363.2535, found 363.2526; [α]D : +3.7 (c = 0.6, CH2Cl2);

for NMR analysis and comparison to the authentic sample see supporting information; Rf (PE/EE 5:1): 0.47.

9-(Z)-Jerangolid E (22). A solution of lactone 6 (36.8 mg, 202 µmol, 3 equiv.) in C7F8 (150 µL) was added to alkene 21 (15.0 mg, 67.0 µmol, 1.00 equiv.). The solvent was degassed by bubbling argon through it for 5 min. Grela catalyst (1.7 mg, 2.5 µmol, 0.04 equiv.) was added, the reaction flask sealed with a glass stopper and heated to 70 °C. After 2 h the reaction was cooled down to room temperature, a second portion of Grela catalyst (5.0 mg, 7.4 µmol, 0.11 equiv.) added and the stirring continued for 2 h at 70 °C. The reaction mixture was cooled down to room temperature and flash chromatography (PE/EE 5:1) of the mixture yielded 9-(Z)-jerangolid E (22) (19.6 mg, 54.0 µmol, crude yield 80 %). The pure product was obtained by semipreparative RPHPLC purification in several batches (average recovery: 95%, isolated yield: 76%). RP-HPLC purification column: Kinetex C18 5 µm, 100 Å; gradient (H2O/MeOH): 95/5-60/40-25/75-5/95, 5 min-10 min-24 min-30 min, flow: 20 ml/min; 1H NMR (500 MHz, CDCl3): δ 5.85 (ddd, J = 15.5, 6.1, 1.1 Hz, 1 H, 7-CH), 5.61 (ddd, J = 15.6, 6.7, 1.5 Hz, 1 H, 6-CH), 5.00 (ddq, J = 9.5, 1.9, 0.9 Hz, 1 H, 9-CH), 4.80 (ddd, J = 11.3, 6.7, 4.4 Hz, 1 H, 5CH), 4.14 (dd, J = 11.5, 2.2 Hz, 1 H, 11-CH), 3.84 (s, 3 H, OMe), 3.30 (m, 1 H, 8-CH), 2.93 (ddd, J = 9.3, 7.3, 2.8 Hz, 1 H, 15-CH), 2.84 (ddq, J = 17.3, 4.1, 1.0 Hz, 1 H, 4-CH2), 2.63 (ddq, J = 17.4, 11.5, 1.9 Hz, 1 H, 4-CH2), 1.81 (dddd, J = 12.5, 3.3, 3.3, 3.3, 1 H, 13-CH2), 1.74-1.65 (m, 1 H, 16-CH2), 1.71 (dd, J = 1.2, 2.1 Hz, 3 H, 18-CH3), 1.70 (d, J = 1.7 Hz, 3 H, 21-CH3), 1.65-1.56 (m, 1 H, 12-CH2), 1.48-1.38 (m, 2 H, 12-CH2, 16-CH2), 1.38-1.32 (m, 1 H, 14-CH), 1.39-1.23 (m, 1 H, 13-CH2), 1.05 (d, J = 6.9 Hz, 3 H, 20-CH3), 0.93 (dd, J = 7.3, 7.3 Hz, 3 H, 17-CH3), 0.84 (d, J = 6.4 Hz, 3 H, 22-CH3) ppm; 13C NMR (125 MHz, CDCl3): δ 171.4 (q, 1-C), 169.4 (q, 3-C), 140.7 (t, 7-CH), 137.8 (q, 10-C), 130.5 (t, 9-CH), 126.5 (t, 6-CH), 102.6 (q, 2-C), 85.5 (t, 15-CH), 78.1 (t, 11-CH), 77.0 (t, 5-CH), 56.3 (p, 19-CH3), 35.5 (t, 8-CH), 35.2 (t, 14-CH), 34.1 (s, 13-CH2), 31.8 (s, 12-CH2), 30.3 (s, 4-CH2), 26.8 (s, 16-CH2), 21.3 (p, 20-CH3), 19.5 (p, 21-CH3), 18.1 (p, 22CH3), 9.7 (p, 17-CH3), 8.8 (p, 18-CH3) ppm; HRMS [ESI] m/z for C22H34O4Na [M+Na]+: calcd 385.2349, found 385.2351; [α]D20: +3.8 (c = 0.3, CH2Cl2); Rf (PE/EE 5:1): 0.37.

Ethyl-(5R,6E,8R,9E)-10-((5S,6R)-6-ethyl-5-methyltetrahydro-2H-pyran-2-yl)-5-hydroxy-2,8-dimethyl-3-oxoundeca-6,9dienoate (24). Alkene 20 (18 mg, 80 µmol, 1.00 equiv.) and 3oxo-5-hydroxyester 10 (43 mg, 200 µmol, 2.5 equiv.) were dissolved in C7F8 (180 µL) dissolved. The solvent was degassed by bubbling argon through it for 5 min. Grela catalyst (4.5 mg, 6.7 µmol, 0.08 equiv.) was added, the reaction flask sealed with a glass stopper and heated to 62 °C. After 2 h the reaction was cooled down to room temperature, a second portion of Grela catalyst (2.7 mg, 4.0 µmol, 0.05 equiv.) added and the stirring continued for 2 h at 62 °C. The reaction mixture was cooled down to room temperature and flash chromatography (pentane/Et2O/EE 8:1:1→6:1:1) yielded 24 (27 mg, 68 µmol, 86 %) as a mixture of the C-2 epimers. 1H NMR (500 MHz, CDCl3): δ 5.65 (ddd, J = 15.4, 6.2, 1.1 Hz, 1 H, 7-CH), 5.41, 5.40 (ddd, J = 15.5, 6.4, 1.6 Hz, 1 H, 6-CH), 5.21 (ddq, J = 8.9, 1.2, 1.3 Hz, 1 H, 9-



CH), 4.55 (m, 1 H, 5-CH), 4.20, 4.20 (q, J = 7.07 Hz, 2 H, OEt), 3.59 (bd, J = 11.1 Hz, 1 H, 11-CH), 3.53, 3.53 (q, J = 7.07 Hz, 1 H, 2-CH), 3.08 (m, 1 H, 8-CH), 2.90 (ddd, J = 9.8, 7.4, 2.7 Hz, 1 H, 15-CH), 2.82-2.68 (m, 2 H, 4-CH2), 1.79 (m, 1 H, 13-CH2), 1.73-1.65 (m, 1 H, 16-CH2), 1.63 (d, J = 1.6 Hz, 3 H, 20-CH3), 1.62 (m, 1 H, 12-CH2), 1.46-1.37 (m, 2 H, 12-CH2, 16-CH2), 1.36-1.32 (m, 4 H, 14-CH, 18-CH3), 1.27 (t, J = 7.1 Hz, 3 H, OEt), 1.27-1.17 (m, 1 H, 13-CH2), 1.05 (d, J = 6.9 Hz, 3 H, 19-CH3), 0.95 (dd, J = 7.4, 7.4 Hz, 3 H, 17-CH3), 0.81 (d, J = 6.6 Hz, 3 H, 21-CH3) ppm; 13C NMR (125 MHz, CDCl3): δ 206.3 (q, 3-C), 170.3 (q, 1-C), 136.7, 136.7 (t, 7-CH), 136.4 (q, 10-C), 128.4, 128.3 (t, 6-CH), 127.9 (t, 9-CH), 84.5 (t, 15-CH), 82.0 (t, 11-CH), 68.8, 68.7 (t, 5-CH), 61.6 (s, OEt), 53.7, 53.6 (t, 2-CH), 48.4, 48.2 (s, 4CH2), 34.6 (t, 8-CH), 34.4 (t, 14-CH), 33.3 (s, 13-CH2), 30.9 (s, 12CH2), 26.0 (s, 16-CH2), 20.8 (p, 19-CH3), 17.9 (p, 21-CH3), 14.2 (p, OEt), 13.3 (p, 20-CH3), 12.7, 12.6 (p, 18-CH3), 9.6 (p, 17-CH3) ppm; HRMS [ESI] m/z for C23H38O5Na [M+Na]+: calcd 417.2611, found 417.2600; Rf (PE/EE 4:1): 0.30.

Projerangolid (3). To a solution of 3-oxo-5-hydroxyester 24 (10.0 mg, 25.3 µmol, 1.0 equiv.) in MeOH (535 µL) was added freshly pestled K2CO3 (7.7 mg, 55.7 µmol, 2.2 equiv.) at 0 °C and the reaction stirred at room temperature for 20 min. The reaction was quenched by addition of a few drops of 0.1 M HCl. The mixture was carefully concentrated under reduced pressure at 30 °C. The residue was taken up in a small amount of EtOAc and 0.05 M HCl was carefully added dropwise until a clear, colorless solution was obtained. The solvent was removed under reduced pressure. The crude product was purified via flash chromatography using reverse phase silica (C8-RP, 12%C, 40-63 µm, Acros Organics; MeOH/H2O+0.001% TFA, gradient: 60-80% MeOH) yielding projerangolid (3) (7.0 mg, 20.0 µmol, 79%) as a mixture of the C2 epimers (see note below). Note: Due to the thermolability and general sensitivity of projerangolid (3), this compound needed to be handled carefully to avoid decomposition. The latter is recognizable by the formation of an orange discoloration after standing at room temperature. Purification accompanied by minimal decomposition was successful using reverse phase flash chromatography. Neither normal phase flash chromatography nor RP HPLC yielded relevant amounts of the pure target compound. As described before for this type of compounds, the 3-methyldihydro-2H-pyran-2,4(3H)-dione exists as a mixture of C-2 epimers in a ratio of approximately 5.3:1 (determined from the intensity ratio of the 2-Hs), resulting in a second set of NMR signals.17 Only the signals for the main stereoisomer are evaluated below. The signal sets of the minor epimer as well as collapsing sets with the main epimer are highlighted with asterisks in the 1H NMR spectrum in the supporting information. 1H NMR (500 MHz, CDCl3): δ 5.85 (ddd, J = 15.5, 6.1, 0.7 Hz, 1 H, 7-CH), 5.49 (ddd, J = 15.5, 6.8, 1.4 Hz, 1 H, 6-CH), 5.22 (m, 1 H, 9-CH), 5.14 (m, 1 H, 5-CH), 3.60 (bd, J = 11.2 Hz, 1 H, 11-CH), 3.57 (q, J = 6.5 Hz, 1 H, 2-CH), 3.15 (m, 1 H, 8-CH), 2.90 (ddd, J = 9.8, 7.4, 2.6 Hz, 1 H, 15-CH), 2.76 (dd, J = 18.8, 3.1 Hz, 1 H, 4-CH2), 2.55 (dd, J = 18.9, 11.7 Hz, 1 H, 4-CH2), 1.80 (m, 1 H, 13-CH2), 1.74-1.66 (m, 1 H, 16-CH2), 1.64 (d, J = 1.4 Hz, 3 H, 20-CH3), 1.67-1.61 (m, 1 H, 12CH2), 1.46-1.38 (m, 2 H, 12-CH2, 16-CH2), 1.37 (d, J = 6.6 Hz, 3 H, 18-CH3), 1.39-1.32 (m, 1 H, 14-CH), 1.25-1.18 (m, 1 H, 13CH2), 1.09 (d, J = 6.9 Hz, 3 H, 19-CH3), 0.95 (dd, J = 7.4, 7.4 Hz, 3 H, 17-CH3), 0.81 (d, J = 6.6 Hz, 3 H, 21-CH3) ppm; 13C NMR (125 MHz, CDCl3): δ 201.4 (q, 3-C), 169.8 (q, 1-C), 140.8 (t, 7-

Page 10 of 13

CH), 137.4 (q, 10-C), 126.8 (t, 9-CH), 123.1 (t, 6-CH), 84.5 (t, 15CH), 81.8 (t, 11-CH), 74.8 (t, 5-CH), 51.9 (t, 2-CH), 43.5 (s, 4CH2), 34.7 (t, 8-CH), 34.4 (t, 14-CH), 33.3 (s, 13-CH2), 31.0 (s, 12CH2), 26.0 (s, 16-CH2), 20.5 (p, 19-CH3), 17.9 (p, 21-CH3), 13.4 (p, 20-CH3), 9.6 (p, 17-CH3), 8.0 (p, 18-CH3) ppm; HRMS [ESI] m/z for C21H32O4Na [M+Na]+: calcd 371.2193, found 371.2195, C21H33O4 [M+H]+: calcd 349.2373, found 349.2376; Rf (PE/EE 2:1): 0.68.

Ethyl-(5S,6E,8R,9E)-10-((5S,6R)-6-ethyl-5-methyltetrahydro-2H-pyran-2-yl)-5-hydroxy-2,8-dimethyl-3-oxoundeca-6,9dienoate (S20). Alkene 20 (15.0 mg, 67.0 µmol, 1.00 equiv.) and β-ketoester 5-epi-10 (67.0 mg, 313 µmol, 4.67 equiv.) were dissolved in C7F8 (180 µL) dissolved. The solvent was degassed by bubbling argon through it for 5 min. Grela-catalyst (2.6 mg, 3.9 µmol, 0.06 equiv.) was added, the reaction flask sealed with a glass stopper and heated to 70 °C. After 2 h the reaction was cooled down to room temperature, a second portion of Grela-catalyst (3.9 mg, 5.8 µmol, 0.09 equiv.) added and the stirring continued for 2 h at 70°C. The reaction mixture was cooled down to room temperature and flash chromatography (PE/EE 5:1) yielded product S20 (20.0 mg, 50.0 µmol, 75 %) as a mixture of the C-2 epimers. 1H NMR (500 MHz, CDCl3): δ 5.64 (m, 1 H, 7-CH), 5.41, 5.40 (ddd, J = 15.5, 6.5, 1.4 Hz, 1 H, 6-CH), 5.21 (m, 1 H, 9-CH), 4.54 (m, 1 H, 5-CH), 4.19 (q, J = 7.1 Hz, 2 H, OEt), 3.59 (bd, J = 11.1 Hz, 1 H, 11-CH), 3.53, 3.52 (q, J = 7.2 Hz, 1 H, 2-CH), 3.08 (m, 1 H, 8-CH), 2.90 (ddd, J = 9.7, 7.4, 2.6 Hz, 1 H, 15-CH), 2.81-2.68 (m, 2 H, 4-CH2), 1.79 (m, 1 H, 13-CH2), 1.73-1.65 (m, 1 H, 16-CH2), 1.63 (d, J = 1.2 Hz, 3 H, 20-CH3), 1.62 (m, 1 H, 12CH2), 1.46-1.38 (m, 2 H, 12-CH2, 16-CH2), 1.36-1.32 (m, 4 H, 14CH, 18-CH3), 1.27 (t, J = 7.2 Hz, 3 H, OEt), 1.27-1.17 (m, 1 H, 13CH2), 1.05 (d, J = 6.9 Hz, 3 H, 19-CH3), 0.95 (dd, J = 7.4, 7.4 Hz, 3 H, 17-CH3), 0.81 (d, J = 6.7 Hz, 3 H, 21-CH3) ppm; 13C NMR (125 MHz, CDCl3): δ 206.4 (q, 3-C), 170.4, 170.3 (q, 1-C), 136.8, 136.7 (t, 7-CH), 136.4 (q, 10-C), 128.4, 128.4 (t, 6-CH), 127.9 (t, 9-CH), 84.5 (t, 15-CH), 81.9 (t, 11-CH), 68.8, 68.7 (t, 5-CH), 61.7 (s, OEt), 53.7, 53.6 (t, 2-CH), 48.4, 48.2 (s, 4-CH2), 34.7 (t, 8-CH), 34.4 (t, 14-CH), 33.3 (s, 13-CH2), 30.9 (s, 12-CH2), 26.0 (s, 16CH2), 20.8 (p, 19-CH3), 17.9 (p, 21-CH3), 14.2 (p, OEt), 13.3 (p, 20-CH3), 12.7, 12.6 (p, 18-CH3), 9.6 (p, 17-CH3) ppm; HRMS [ESI] m/z for C23H38O5Na [M+Na]+: calcd 471.2611, found 417.2600; Rf (PE/EE 5:1): 0.44.

5-epi-Projerangolid (25). To a solution of 3-oxo-5-hydroxyester S20 (9.3 mg, 24 µmol, 1.0 equiv.) in MeOH (450 µL) was added freshly pestled K2CO3 (6.5 mg, 47 µmol, 2 equiv.) and the reaction stirred at room temperature for 30 min. The mixture was concentrated under reduced pressure. The residue was taken up in a small amount of EtOAc and 0.05 M HCl was carefully added dropwise until a clear, colorless solution was obtained. The aqueous layer was extracted three times with EtOAc. The combined organic layers were dried over NaSO4 and concentration under reduced pressure gave 5-epi-Projerangolid (25) (6.2 mg, 18.0 µmol, 75 %) as a mixture of the C-2 epimers in sufficient purity for enzymatic bioconversion. 1H NMR (500 MHz, CDCl3): δ 5.84 (ddd, J = 15.4, 6.3, 0.8 Hz, 1 H, 7-CH), 5.48 (ddd, J = 15.5, 6.8, 1.5 Hz, 1 H, 6-CH), 5.22 (m, 1 H, 9-CH), 5.14 (m, 1 H, 5-CH), 3.60 (bd,


Page 11 of 13 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


J = 11.0 Hz, 1 H, 11-CH), 3.57 (q, J = 6.5 Hz, 1 H, 2-CH), 3.15 (m, 1 H, 8-CH), 2.90 (ddd, J = 9.4, 7.7, 2.4 Hz, 1 H, 15-CH), 2.76 (dd, J = 19.1, 2.9 Hz, 1 H, 4-CH2), 2.55 (dd, J = 19.0, 11.7 Hz, 1 H, 4-CH2), 1.79 (m, 1 H, 13-CH2), 1.74-1.66 (m, 1 H, 16-CH2), 1.64 (d, J = 1.4 Hz, 3 H, 20-CH3), 1.67-1.61 (m, 1 H, 12-CH2), 1.46-1.38 (m, 2 H, 12-CH2, 16-CH2), 1.37 (d, J = 6.7 Hz, 3 H, 18-CH3), 1.39-1.32 (m, 1 H, 14-CH), 1.25-1.18 (m, 1 H, 13-CH2), 1.09 (d, J = 6.9 Hz, 3 H, 19-CH3), 0.95 (dd, J = 7.4, 7.4 Hz, 3 H, 17-CH3), 0.81 (d, J = 6.6 Hz, 3 H, 21-CH3) ppm; 13C NMR (125 MHz, CDCl3): δ 201.4 (q, 3-C), 169.8 (q, 1-C), 141.0 (t, 7CH), 137.3 (q, 10-C), 126.8 (t, 9-CH), 123.1 (t, 6-CH), 84.5 (t, 15CH), 81.7 (t, 11-CH), 74.9 (t, 5-CH), 51.9 (t, 2-CH), 43.5 (s, 4CH2), 34.8 (t, 8-CH), 34.4 (t, 14-CH), 33.3 (s, 13-CH2), 31.0 (s, 12CH2), 26.0 (s, 16-CH2), 20.6 (p, 19-CH3), 17.9 (p, 21-CH3), 13.5 (p, 20-CH3), 9.6 (p, 17-CH3), 8.0 (p, 18-CH3) ppm; HRMS [ESI] m/z for C21H32O4Na [M+Na]+: calcd 371.2193, found 371.2188, C21H33O4 [M+H]+: calcd 349.2373, found 349.2368; Rf (PE/EE 3:1): 0.29. Enzyme expression. Recombinant plasmid jerF_pCOLD-I was used to transform E. coli BL21 (DE3) cells. The resulting transformants were used to generate overnight cultures. The following day, these cultures were used to inoculate 50 mL medium in 250 mL-flasks to an initial OD600 of 0.05. The cultures were grown at 37 °C, 180 rpm for 2-3 h until OD600 reached 0.6-0.8. Isopropylβ-D-thiogalactopyranoside (IPTG) was added to a final concentration of 0.1 mM to induce gene expression. The cells were cultured at 16 °C for 21 h. Cells were harvested by centrifugation (3200 g, 4 °C, 30 min) and cell disruption was carried out by sonication of 1 g cells resuspended in 10 mL in assay buffer (25 mM HEPES, 100 mM NaCl, pH 7.5). After centrifugation (10000 g, 4 °C, 45 min) the cell free extract was used directly for bioconversion. Bioconversion experiments of JerF with 3 and 25. The experiments were carried out in 200 µL of reaction buffer (25 mM HEPES, 100 mM NaCl, 5 mM MgCl2, pH 7.5) containing JerF (1.6 mg/mL total protein), 0.25 mM projerangolid and 2.92 mM SAM-tosylate as methyl donor. For assays with cosolvent 0.5 % MeOH or DMSO were added. The assays were incubated at 28 °C for 20 h before 100 µL of brine were added. The aqueous layer was extracted two times with EtOAc (200 µL). The combined organic extracts were dried, redissolved in 200 µL MeOH and analyzed via UPLC-MS. For time course experiments, different amounts of JerF (1.90, 0.95 and 0.48 mg/mL total protein) were applied in the assays under the above-mentioned conditions in the presence of 0.5 % DMSO cosolvent. After workup, the results were analyzed by UPLC-MS. Reactions were stopped after 2, 6, 12, 22, 32, 48, 64 and 94 minutes (double determination). For the investigation on solvent tolerance, varying amounts of DMSO (0.5, 1, 2, 5, 10, 15, 20, 50 vol%) were applied and the total volume of the assays filled up to 200 µL (2.3 mg/mL total protein, 1.0 mM SAM-tosylate). After workup, the results were analyzed by UPLC-MS.

ASSOCIATED CONTENT

Frank Hahn: 0000-0002-0603-3482

Present Address †Zentrum für Biomolekulare Wirkstoffe Leibniz Universität Hannover Schneiderberg 38, 30167 Hannover (Germany)

Notes The authors declare no competing financial interests.

ACKNOWLEDGMENT Funding from the Emmy Noether program of the DFG (HA 5841/2-1) and the Marie Curie program of the European Union (project number 293430) is gratefully acknowledged.

REFERENCES (1)

(2)

(3)

(4) (5)

(6)

(7) (8) (9)

(10)

(11)

Supporting Information The Supporting Information is available free of charge on the ACS Publications website. full synthetic scheme, NMR spectra, HRMS analysis, LC-MS analysis (PDF)

AUTHOR INFORMATION Corresponding Author * E-Mail: [email protected]

ORCID:

(12)

(13)

Gerth, K.; Washausen, P.; Hofle, G.; Irschik, H.; Reichenbach, H. The Jerangolids: A Family of New Antifungal Compounds from Sorangium Cellulosum (Myxobacteria). Production, Physico-Chemical and Biological Properties of Jerangolid A. J. Antibiot. 1996, 49 (1), 71–75. Knauth, P.; Reichenbach, H. On the Mechanism of Action of the Myxobacterial Fungicide Ambruticin. J. Antibiot. 2000, 53 (10), 1182–1190. Kojima, K.; Takano, Y.; Yoshimi, A.; Tanaka, C.; Kikuchi, T.; Okuno, T. Fungicide Activity through Activation of a Fungal Signalling Pathway. Molecular Microbiology 53 (6), 1785–1796. Ringel, S. M. New Antifungal Agents for the Systemic Mycoses. Mycopathologia 1990, 109 (2), 75–87. Vetcher, L.; Menzella, H. G.; Kudo, T.; Motoyama, T.; Katz, L. The Antifungal Polyketide Ambruticin Targets the HOG Pathway. Antimicrob. Agents Chemother. 2007, 51 (10), 3734–3736. Dongo, A.; Bataillé-Simoneau, N.; Campion, C.; Guillemette, T.; Hamon, B.; Iacomi-Vasilescu, B.; Katz, L.; Simoneau, P. The Group III Two-Component Histidine Kinase of Filamentous Fungi Is Involved in the Fungicidal Activity of the Bacterial Polyketide Ambruticin. Appl. Environ. Microbiol. 2009, 75 (1), 127–134. Pospíšil, J.; Markó, I. E. Total Synthesis of Jerangolid D. J. Am. Chem. Soc. 2007, 129 (12), 3516–3517. Hanessian, S.; Focken, T.; Oza, R. Total Synthesis of Jerangolid A. Org. Lett. 2010, 12 (14), 3172–3175. Hanessian, S.; Focken, T.; Mi, X.; Oza, R.; Chen, B.; Ritson, D.; Beaudegnies, R. Total Synthesis of (+)-Ambruticin S: Probing the Pharmacophoric Subunit. J. Org. Chem. 2010, 75 (16), 5601–5618. Berberich, S. M.; Cherney, R. J.; Colucci, J.; Courillon, C.; Geraci, L. S.; Kirkland, T. A.; Marx, M. A.; Schneider, M. F.; Martin, S. F. Total Synthesis of (+)-Ambruticin S. Tetrahedron 2003, 59 (35), 6819–6832. Tian, Z.-Q.; Wang, Z.; Xu, Y.; Tran, C. Q.; Myles, D. C.; Zhong, Z.; Simmons, J.; Vetcher, L.; Katz, L.; Li, Y.; et al. Investigating Amine Derivatives of Ambruticin VS-5 and VS-4. ChemMedChem 2008, 3 (6), 963–969. Xu, Y.; Wang, Z.; Tian, Z.-Q.; Li, Y.; Shaw, S. J. Investigating Carboxylic Acid Analogues of Ambruticin through Semi-Synthesis. ChemMedChem 2006, 1 (10), 1063– 1065. Julien, B.; Tian, Z.-Q.; Reid, R.; Reeves, C. D. Analysis of the Ambruticin and Jerangolid Gene Clusters of Sorangium Cellulosum Reveals Unusual Mechanisms of Polyketide Biosynthesis. Chemistry & Biology 2006, 13 (12), 1277–1286.



(14)

(15)

(16)

(17)

(18)

(19)

(20)

(21)

(22)

(23)

(24)

Berkhan, G.; Hahn, F. A Dehydratase Domain in Ambruticin Biosynthesis Displays Additional Activity as a Pyran-Forming Cyclase. Angew. Chem. Int. Ed. 2014, 53 (51), 14240–14244. Berkhan, G.; Merten, C.; Holec, C.; Hahn, F. The Interplay between a Multifunctional Dehydratase Domain and a CMethyltransferase Effects Olefin Shift in Ambruticin Biosynthesis. Angew. Chem. Int. Ed. 2016, 55 (43), 13589– 13592. Sung, K. H.; Berkhan, G.; Hollmann, T.; Wagner, L.; Blankenfeldt, W.; Hahn, F. Insights into the Dual Activity of a Bifunctional Dehydratase-Cyclase Domain. Angew. Chem. Int. Ed. 2018, 57 (1), 343–347. Friedrich, S.; Hemmerling, F.; Lindner, F.; Warnke, A.; Wunderlich, J.; Berkhan, G.; Hahn, F. Characterisation of the Broadly-Specific O-Methyl-Transferase JerF from the Late Stages of Jerangolid Biosynthesis. Molecules 2016, 21 (11), 1443. Minami, A.; Migita, A.; Inada, D.; Hotta, K.; Watanabe, K.; Oguri, H.; Oikawa, H. Enzymatic Epoxide-Opening Cascades Catalyzed by a Pair of Epoxide Hydrolases in the Ionophore Polyether Biosynthesis. Org. Lett. 2011, 13 (7), 1638–1641. Minami, A.; Shimaya, M.; Suzuki, G.; Migita, A.; Shinde, S. S.; Sato, K.; Watanabe, K.; Tamura, T.; Oguri, H.; Oikawa, H. Sequential Enzymatic Epoxidation Involved in Polyether Lasalocid Biosynthesis. J. Am. Chem. Soc. 2012, 134 (17), 7246–7249. Gallimore, A. R.; Stark, C. B. W.; Bhatt, A.; Harvey, B. M.; Demydchuk, Y.; Bolanos-Garcia, V.; Fowler, D. J.; Staunton, J.; Leadlay, P. F.; Spencer, J. B. Evidence for the Role of the MonB Genes in Polyether Ring Formation during Monensin Biosynthesis. Chemistry & Biology 2006, 13 (4), 453–460. Smith, T. E.; Djang, M.; Velander, A. J.; Downey, C. W.; Carroll, K. A.; van Alphen, S. Versatile Asymmetric Synthesis of the Kavalactones: First Synthesis of (+)-Kavain. Org. Lett. 2004, 6 (14), 2317–2320. Feng, Y.-S.; Wu, W.; Xu, Z.-Q.; Li, Y.; Li, M.; Xu, H.-J. Pd-Catalyzed Decarboxylative Cross-Couplings of Potassium Malonate Monoesters with Aryl Halides. Tetrahedron 2012, 68 (9), 2113–2120. Bates, R. W.; Li, L.; Palani, K.; Phetsang, W.; Loh, J. K. Synthesis of the Tetrahydropyran Fragment of Bistramide D. Asian Journal of Organic Chemistry 2014, 3 (7), 792– 796. Wünsch, S.; Breit, B. Probing O-Diphenylphosphanyl Benzoate (o-DPPB)-Directed C C Bond Formation:

(25)

(26)

(27)

(28)

(29)

(30)

(31)

(32)

(33)

(34)

Page 12 of 13

Total Synthesis of Dictyostatin. Chemistry – A European Journal 2015, 21 (6), 2358–2363. Jasper, C.; Wittenberg, R.; Quitschalle, M.; Jakupovic, J.; Kirschning, A. Total Synthesis and Elucidation of the Absolute Configuration of the Diterpene Tonantzitlolone †. Organic Letters 2005, 7 (3), 479–482. Naruta, Y.; Nishigaichi, Y.; Maruyama, K. Optically Active Pentadienyltin Reagent and Its Application to Asymmetric Synthesis of (R)-7,11-Dideoxydaunomycinone. J. Chem. Soc., Chem. Commun. 1989, 0 (17), 1203–1205. Ishigai, K.; Fuwa, H.; Hashizume, K.; Fukazawa, R.; Cho, Y.; Yotsu‐Yamashita, M.; Sasaki, M. Total Synthesis and Biological Evaluation of (+)-Gambieric Acid A and Its Analogues. Chemistry – A European Journal 2013, 19 (17), 5276–5288. Tsubone, K.; Hashizume, K.; Fuwa, H.; Sasaki, M. Studies toward the Total Synthesis of Gambieric Acids: Convergent Synthesis of the GHIJ-Ring Fragment Having a Side Chain. Tetrahedron Letters 2011, 52 (4), 548–551. Chatterjee, A. K.; Choi, T.-L.; Sanders, D. P.; Grubbs, R. H. A General Model for Selectivity in Olefin Cross Metathesis. J. Am. Chem. Soc. 2003, 125 (37), 11360–11370. Chatterjee, A. K.; Morgan, J. P.; Scholl, M.; Grubbs, R. H. Synthesis of Functionalized Olefins by Cross and RingClosing Metatheses. J. Am. Chem. Soc. 2000, 122 (15), 3783–3784. Samojłowicz, C.; Bieniek, M.; Pazio, A.; Makal, A.; Woźniak, K.; Poater, A.; Cavallo, L.; Wójcik, J.; Zdanowski, K.; Grela, K. The Doping Effect of Fluorinated Aromatic Solvents on the Rate of Ruthenium-Catalysed Olefin Metathesis. Chemistry – A European Journal 2011, 17 (46), 12981–12993. Grela, K.; Harutyunyan, S.; Michrowska, A. A Highly Efficient Ruthenium Catalyst for Metathesis Reactions. Angewandte Chemie International Edition 2002, 41 (21), 4038–4040. Oppolzer, W.; Barras, J.-P. Asymmetric Dihydroxylations of ?-SubstitutedN-(?,?-Enoyl)bornane-10,2-sultams. Helvetica Chimica Acta 1987, 70 (7), 1666–1675. Wünsch, S.; Breit, B. Probing o -Diphenylphosphanyl Benzoate ( o -DPPB)-Directed C C Bond Formation: Total Synthesis of Dictyostatin. Chemistry - A European Journal 2015, 21 (6), 2358–2363.


Page 13 of 13 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Table of contents


Total Synthesis of Complex Biosynthetic Late ... - ACS Publications

Recommend Documents