Total Syntheses of the Proposed Structure of Iriomoteolide-1a, -1b and Synthesis of Three Derivatives for Structural Studies

Iriomoteolide-1a and iriomoteolide-1b are very potent cytotoxic agents, isolated from marine dinoflagellates. We carried out the enantioselective syntheses of the proposed structures of these natural products. However, our analysis of the NMR spectra of the synthetic iriomoteolide-1a and the natural products revealed that the structures of iriomoteolide-1a and iriomoteolide-1b were assigned incorrectly. Based upon our detailed analysis of the spectral data of the synthetic iriomoteolide-1a and the natural products, we rationally designed three diastereomers of the proposed structure of 1 in an effort to assign the correct structures. The key steps of our syntheses of the proposed structures of iriomoteolides involved a highly diastereoselective ene reaction, a carbocupration that utilized a Gilman reagent, a Julia–Kocienski olefination to couple fragments, and Yamaguchi macrolactonization to form the target macrolactone. This synthetic route was then utilized to carry out syntheses of three diastereomers to the proposed structure of 1. These diastereomeric structures show close similarities to natural iriomoteolide-1a; however, there were differences in their spectral data. While natural iriomoteolides exhibited potent cytotoxicies, our preliminary biological evaluation of synthetic iriomoteolide-1a, iriomoteolide-1b, and all three synthetic derivatives did not show any appreciable cytotoxic properties.


Introduction
Marine natural products are a great source of structurally intriguing bioactive molecules with novel modes of action [1,2]. The field of marine natural products is immensely important in modern drug discovery. Already, many new approved drugs with interesting biological mechanisms are in pharmacies [3,4]. The field has great potential in modern medicine; however, it is vastly unexplored. The synthesis of these bioactive molecules and exploration of structure activity relationship studies are playing an important role in drug discovery today [5,6]. Iriomoteolide-1a (1) (Figure 1) is a 20-memembered cytotoxic macrolide, which was isolated by Tsuda and co-workers from a benthic HYA024 strain of dinoflagellate Amphidinium sp. collected off Iriomote Island, Japan in 2007 [7,8]. It displayed very potent cytotoxicity against human B lymphocyte DG-75 cells, with an IC 50 value of 2 ng/mL. Furthermore, it exhibited cytotoxicity against Epstein-Barr virus (EBV)-infected human lymphocyte Raji cells (IC 50 = 3 ng/mL) [7,8]. The initial structure of iriomoteolide-1a (1) was determined based on extensive 2D-NMR studies and mass spectroscopic analyses. The relative and absolute configurations were assigned based on the NMR studies, conformational analyses of derivatives of 1 with Mosher's reagent. Later, Tsuda et al. reported the isolation of iriomoteolide-1b (2) [8], which was isolated from the same HY A024 strain of dinoflagellate Amphidinium sp. Iriomoteolide-1b (2) is structurally related to iriomoteolide-1a (1). Instead of a 6-membered hemiketal ring at the C9-C13 position and an exo-methylene group at C11 in iriomoteolide-1a (1), iriomoteolide-1b (2) possesses a ketone at C13 conjugated with a Z-double bond at C11-C12 and a hydroxyl group at C9. Treatment of iromoteolide-1a (1) with triethylamine in dichloromethane for 168 h furnished a polar product. The 1 H NMR analyses reveal that the product is identical to iriomoteolide-1b (2). However, the IC 50 value of 2 against DG-75 cells is found to be less potent than that of 1 (IC 50 900 ng/mL) [7,8]. Iriomoteolides targets have attracted considerable synthetic interest, leading to the syntheses of various segments of iriomoteolides [9][10][11][12][13][14][15][16][17]. In addition, synthesis of the proposed structures of iriomoteolides, as well as syntheses of structural variants of iriomoteolides, have been reported [18][19][20][21][22][23]. Thus far, neither the biological mechanism of action nor the correct structures of iriomotelides have been reported. Herein, we report our revised syntheses of the proposed structures of iriomoteolide-1a and -1b. Our convergent and highly stereoselective synthetic route was utilized for the syntheses of three rationally designed structural variants for structural elucidation and biological studies.

Synthetic Plan
Our retrosynthetic analysis is outlined in Figure 2. Our convergent synthetic strategy involves a Julia-Kocienski olefination [24,25] of aldehyde 3 and sulfone 4. The resulting trans-olefin intermediate was converted to iriomoteolide macrolatone, using Yamaguchi macrolactonization [26] as the key step to build the 20-membered macrolactone. The synthesis of C1-C15 fragment 3 relied upon another Julia-Kocienski olefination from sulfone 5 and aldehyde 6. An ene reaction of aldehyde 7 and olefin 8 was designed to furnish Sulfone 5. A Cu(I)-mediated epoxide ring opening reaction provides the olefin 8 from expoxide 9, which was readily prepared from the known alcohol 10. The synthesis of C16-C23 segment 4 was planned from alkene 11 by hydroboration-oxidation, followed by conversion of the resulting alcohol to sulfone derivative 4. Alkene 11 was be synthesized using an asymmetric crotylboration of the aldehyde derived from 12 as the key step. Optically active alcohol 12 can be conveniently obtained from asymmetric crotylboration of acetaldehyde. Iriomoteolide-1b (2)

Synthetic Plan
Our retrosynthetic analysis is outlined in Figure 2. Our convergent synthetic strategy involves a Julia-Kocienski olefination [24,25] of aldehyde 3 and sulfone 4. The resulting trans-olefin intermediate was converted to iriomoteolide macrolatone, using Yamaguchi macrolactonization [26] as the key step to build the 20-membered macrolactone. The synthesis of C1-C15 fragment 3 relied upon another Julia-Kocienski olefination from sulfone 5 and aldehyde 6. An ene reaction of aldehyde 7 and olefin 8 was designed to furnish Sulfone 5. A Cu(I)-mediated epoxide ring opening reaction provides the olefin 8 from expoxide 9, which was readily prepared from the known alcohol 10. The synthesis of C16-C23 segment 4 was planned from alkene 11 by hydroboration-oxidation, followed by conversion of the resulting alcohol to sulfone derivative 4. Alkene 11 was be synthesized using an asymmetric crotylboration of the aldehyde derived from 12 as the key step. Optically active alcohol 12 can be conveniently obtained from asymmetric crotylboration of acetaldehyde.

Synthesis of C7-C15 Fragment 5
The synthesis of C7-C15 fragment 5 was planned by using a diastereoselective ene reaction [27,28], as outlined in Scheme 1. Treatment of the known alcohol 10 with 1-phenyl-1H-tetrazole-5-thiol under Mitsunobu's condition [29] afforded the corresponding sulfide. Deprotection of the acetonide group gave the diol 13. Tosylation of diol 13 in the presence of triethylamine and dibutyltin oxide, followed by treatment of potassium carbonate in a mixture of methanol and dichloromethane, furnished the epoxide 14. Reaction of epoxide 14 with isopropenylmagnesium bromide in the presence of a catalytic amount of copper(I) cyanide resulted in the corresponding alcohol. Interestingly, Curran and coworkers reported that the reaction of the enantiomer of 14 with an alkynyllithium reagent furnished a by-product that resulted from the displacement of 1-phenyltetrazole [30]. The alcohol from 14 was then protected with TBSCl to afford the silyl ether 8. A SnCl 4 -mediated ene reaction of aldehyde 7 and olefin 8 was carried out to provide the corresponding alcohol in 76% yield as a mixture of diastereomers with good selectivity (8:1 dr). The use of TiCl 4 as a Lewis acid led to the desired product but with a lower yield (40%). A chelation-controlled addition between the α-hydroxyl group and the aldehyde resulted in Mar. Drugs 2022, 20, 587 3 of 30 good diastereoselectivity (8:1 dr) for the ene reaction [31,32]. The diol was protected as an acetonide derivative to afford 15. The absolute configuration of the new chiral center at C13 was identified as drawn in 15 (R-configuration) using 1 H-NMR NOESY experiments. NOESY between H13 and H15, and NOESY between H12 and H27 were observed, as shown in acetonide 15.

Synthesis of C7-C15 Fragment 5
The synthesis of C7-C15 fragment 5 was planned by using a diastereoselective ene reaction [27,28], as outlined in Scheme 1. Treatment of the known alcohol 10 with 1phenyl-1H-tetrazole-5-thiol under Mitsunobu's condition [29] afforded the corresponding sulfide. Deprotection of the acetonide group gave the diol 13. Tosylation of diol 13 in the presence of triethylamine and dibutyltin oxide, followed by treatment of potassium carbonate in a mixture of methanol and dichloromethane, furnished the epoxide 14.
Reaction of epoxide 14 with isopropenylmagnesium bromide in the presence of a catalytic amount of copper(I) cyanide resulted in the corresponding alcohol. Interestingly, Curran and co-workers reported that the reaction of the enantiomer of 14 with an alkynyllithium reagent furnished a by-product that resulted from the displacement of 1-phenyltetrazole [30]. The alcohol from 14 was then protected with TBSCl to afford the silyl ether 8. A SnCl4mediated ene reaction of aldehyde 7 and olefin 8 was carried out to provide the corresponding alcohol in 76% yield as a mixture of diastereomers with good selectivity (8:1 dr). The use of TiCl4 as a Lewis acid led to the desired product but with a lower yield (40%). A chelation-controlled addition between the α-hydroxyl group and the aldehyde resulted in good diastereoselectivity (8:1 dr) for the ene reaction [31,32]. The diol was protected as an acetonide derivative to afford 15. The absolute configuration of the new chiral center at C13 was identified as drawn in 15 (R-configuration) using 1 H-NMR NOESY experiments. NOESY between H13 and H15, and NOESY between H12 and H27 were observed, as shown in acetonide 15. While this new chiral center would be removed in the late stage of the synthesis via oxidation to the corresponding ketone, good diastereoselectivity in the ene reaction simplified the NMR spectra. Sulfide 15 was then oxidized by ammonium molybdate and hydrogen peroxide to afford the sulfone 5 in good yield. While this new chiral center would be removed in the late stage of the synthesis via oxidation to the corresponding ketone, good diastereoselectivity in the ene reaction simplified the NMR spectra. Sulfide 15 was then oxidized by ammonium molybdate and hydrogen peroxide to afford the sulfone 5 in good yield.
2.2.1. Syntheses of C16-C23 Segment 4 and C1-C6 Fragment 6 The synthesis of C16-C23 segment 4 was carried out, as shown in Scheme 2. Asymmetric crotylboration of acetaldehyde using cis-2-butene and (+)-B-methoxy-diisopinocamphen ylborane using the protocol developed by Brown and co-workers [33,34] furnished optically active syn-alcohol 12. As reported previously [19], alcohol functionality was protected as a TBS-ether and hydroboration-oxidation of the alkene under standard condition afforded alcohol 16 in good yield. Swern oxidation of 16 provided the aldehyde, which was subjected to Brown's asymmetric crotylboration using (−)-B-methoxy-diisopinocamphenylborane and trans-2-butene to afford alcohol 17 in good yield and with excellent diastereoselectivity (10:1). Alcohol 17 was protected as a PMB-ether and hydroboration-oxidation of the olefin furnished alcohol 18. Syntheses of derivatives of 17 and 18 with different protecting groups have been reported [17]. This was converted to sulfone 4 by the Mitsunobu reaction, followed by oxidation of the sulfide to sulfone. While this new chiral center would be removed in the late stage of the synthesis via oxidation to the corresponding ketone, good diastereoselectivity in the ene reaction simplified the NMR spectra. Sulfide 15 was then oxidized by ammonium molybdate and hydrogen peroxide to afford the sulfone 5 in good yield.

Syntheses of C16-C23 Segment 4 and C1-C6 Fragment 6
The synthesis of C16-C23 segment 4 was carried out, as shown in Scheme 2 Asymmetric crotylboration of acetaldehyde using cis-2-butene and (+)-B-methoxy diisopinocamphenylborane using the protocol developed by Brown and co-worker [33,34] furnished optically active syn-alcohol 12. As reported previously [19], alcoho functionality was protected as a TBS-ether and hydroboration-oxidation of the alkene under standard condition afforded alcohol 16 in good yield. Swern oxidation of 16 provided the aldehyde, which was subjected to Brown's asymmetric crotylboration using (−)-B-methoxy-diisopinocamphenylborane and trans-2-butene to afford alcohol 17 in good yield and with excellent diastereoselectivity (10:1). Alcohol 17 was protected as a PMB-ether and hydroboration-oxidation of the olefin furnished alcohol 18. Syntheses o derivatives of 17 and 18 with different protecting groups have been reported [17]. Thi was converted to sulfone 4 by the Mitsunobu reaction, followed by oxidation of the sulfide to sulfone. The synthesis of C1-C6 fragment 6 is shown in Scheme 3. The racemic alcohol 19, obtained from the aldol reaction of tert-butylacetate and acrolein, was subjected to immobilized lipase PS-30 catalyzed kinetic resolution in pentane in the presence of excess vinyl acetate, at 30 • C for 19 h, to provide enantio-enriched (R)-19 in 98% ee, along with the corresponding enantiomeric acetate derivative [35]. Treatment of (R)-19 with lithium diisopropylamide, followed by the reaction of the resulting dianion with methyl iodide as described by previously [36], afforded the corresponding anti-alcohol as a single isomer by 1 H NMR analysis. The resulting alcohol was protected as a MOM-ether. Reduction of the resulting ester with LAH furnished alcohol 20. Synthesis of derivative of 20 with different protecting groups was reported [17]. Swern oxidation, followed by Corey-Fuchs' homologation [37] of the aldehyde, provided the corresponding dibromo olefin. Treatment of the dibromide with butyllithium, followed by reaction of the resulting alkynyl anion with methyl chloroformate, furnished alkynyl ester 21 in excellent yield. A carbocupration of alkynyl ester 21 was carried out with freshly prepared Gilman reagent [38] at −40 • C to provide Z-olefin 22 as a single product in excellent isolated yield. The observed NOE between the protons at C2 and Me at C3 is consistent with the assigned Z geometry in ester 22. DIBAL-H reduction of 22, followed by protection of the resulting alcohol with TBSCl, furnished the corresponding silyl ether. Selective oxidative cleavage of the terminal olefin provided the C1-C6 fragment 6. carbocupration of alkynyl ester 21 was carried out with freshly prepared Gilman reagen [38] at −40 °C to provide Z-olefin 22 as a single product in excellent isolated yield. Th observed NOE between the protons at C2 and Me at C3 is consistent with the assigned Z geometry in ester 22. DIBAL-H reduction of 22, followed by protection of the resulting alcohol with TBSCl, furnished the corresponding silyl ether. Selective oxidative cleavag of the terminal olefin provided the C1-C6 fragment 6. Scheme 3. Synthesis of C1-C6 fragment 6.
With sulfone 5 and aldehyde 6 in hand, total syntheses of the proposed structures o iriomoteolide-1a and 1b were successfully achieved. The synthesis featured tw successive Julia-Kocienski olefinations. As shown in Scheme 4, the first Julia-Kociensk reaction between sulfone 5 and aldehyde 6 afforded trans-olefin 23 in excellent yield Removal of the benzyl ether, followed by DMP oxidation [39,40] of the resulting alcoho afforded aldehyde 3. A second Julia-Kocienski reaction of aldehyde 3 and sulfone furnished E-olefin 24 as the only isolated product in good yield. Removal of PMB ether followed by selective removal of the primary TBS-ether with NH4F, resulted in allyli alcohol 25. Oxidation of 25 with MnO2 followed by NaClO2 afforded the correspondin carboxylic acid [41]. Yamaguchi macrolactonization furnished macrolactone 26 in good yield. Macrolactone 26 was converted to the proposed structures of irimoteolide-1a and irimoteolide-1b, as shown in Scheme 5. Treatment of 26 with HF•Py followed by aqueou AcOH resulted in tetraol derivative 27. Bromocatecholborane promoted the removal o the MOM group and furnished the corresponding pentaol derivative. Treatment of th free alcohols with TESCl and DMAP selectively provided TES-ether derivative 28 in good yield. DMP oxidation [39,40] of the secondary alcohol provided the corresponding keton and removal of the TES groups with exposure to HF•Py furnished iriomoteolide-1a (1) in 56% yield and iriomoteolide-1b (2) in 17% yield after silica gel chromatography. The 1 H Scheme 3. Synthesis of C1-C6 fragment 6.
With sulfone 5 and aldehyde 6 in hand, total syntheses of the proposed structures of iriomoteolide-1a and 1b were successfully achieved. The synthesis featured two successive Julia-Kocienski olefinations. As shown in Scheme 4, the first Julia-Kocienski reaction between sulfone 5 and aldehyde 6 afforded trans-olefin 23 in excellent yield. Removal of the benzyl ether, followed by DMP oxidation [39,40] of the resulting alcohol, afforded aldehyde 3. A second Julia-Kocienski reaction of aldehyde 3 and sulfone 4 furnished E-olefin 24 as the only isolated product in good yield. Removal of PMB ether, followed by selective removal of the primary TBS-ether with NH 4 F, resulted in allylic alcohol 25. Oxidation of 25 with MnO 2 followed by NaClO 2 afforded the corresponding carboxylic acid [41]. Yamaguchi macrolactonization furnished macrolactone 26 in good yield. Macrolactone 26 was converted to the proposed structures of irimoteolide-1a and irimoteolide-1b, as shown in Scheme 5. Treatment of 26 with HF•Py followed by aqueous AcOH resulted in tetraol derivative 27. Bromocatecholborane promoted the removal of the MOM group and furnished the corresponding pentaol derivative. Treatment of the free alcohols with TESCl and DMAP selectively provided TES-ether derivative 28 in good yield. DMP oxidation [39,40] of the secondary alcohol provided the corresponding ketone and removal of the TES groups with exposure to HF•Py furnished iriomoteolide-1a (1) in 56% yield and iriomoteolide-1b (2) in 17% yield after silica gel chromatography. The 1 H-NMR and 13 C-NMR of synthetic iriomoteolide-1a and iriomoteolide-1b did not match with the reported data for these natural products [7,8].
Although the 1 H NMR and 13 C NMR spectral data of our synthetic iriomoteolide-1a (1) are comparable to those of independent work reported from other groups [18,20], neither data of 1 nor data of 2 matched those of natural iriomoteolide-1a and -1b (Please see Supplementary Materials for NMR comparison). This suggested that the structures of both natural iriomoteolide-1a and iriomoteolide-1b have been assigned incorrectly. While there are many minor differences, the major discrepancies involve the 1 H and 13 C shifts at C4 (3.98 ppm and 40.6 ppm, respectively, for synthetic iriomoteolide-1a compared to 2.46 ppm and 47.9 ppm, respectively, for the natural product). In addition, there is a distinction of chemical shifts at C24 (1.96 and 20.8 ppm, respectively, for synthetic 1 compared to 2.12 and 23.8 ppm, respectively, for natural 1). These discrepancies reveal that the α,β-unsaturated double bond configuration might be E instead of Z. In addition, the structure may be an epimer at the C4 and C5 positions. Based on the NMR analysis, three diastereomers, 29, 30 and 31 (Figure 3), were designed. The syntheses of these structural variants were carried out utilizing our convergent synthetic route.
Although the 1 H NMR and 13 C NMR spectral data of our synthetic iriomoteolide-1a (1) are comparable to those of independent work reported from other groups [18,20], neither data of 1 nor data of 2 matched those of natural iriomoteolide-1a and -1b (Please see supplementary materials for NMR comparison). This suggested that the structures of ppm and 47.9 ppm, respectively, for the natural product). In addition, there is a distinction of chemical shifts at C24 (1.96 and 20.8 ppm, respectively, for synthetic 1 compared to 2.12 and 23.8 ppm, respectively, for natural 1). These discrepancies reveal that the α,βunsaturated double bond configuration might be E instead of Z. In addition, the structure may be an epimer at the C4 and C5 positions. Based on the NMR analysis, three diastereomers, 29, 30 and 31 (Figure 3), were designed. The syntheses of these structural variants were carried out utilizing our convergent synthetic route.

Synthesis of Diastereomer 29
The synthesis of diastereomer 29 requires alteration of olefin geometry at C2-C3. As shown in Scheme 6, a carbocupration of alkynyl ester 21 with Gilman reagent at 0 °C in the presence of TMSCl resulted in the desired E-olefin as a major isomer (E:Z = 7:3) [38]. DIBAL-H reduction furnished the alcohol 32, which was separated from its Z-isomer by flash column chromatography over silica gel. Protection of alcohol with tertbutyldimethylsilyl chloride and oxidative cleavage of the terminal olefin provided the C1-C6 fragment aldehyde 33 for diastereomer 29. Treatment of sulfone 5 with slightly less than one equivalent of KHMDS, followed by exposure to aldehyde 33, afforded the Eolefin. However, we found that the use of more than one equivalent base led to the epimerization of the chiral center on C5. Removal of the benzyl group followed by Dess-Martin oxidation [39,40] provided the C1-C15 fragment, aldehyde 34. Scheme 6. Synthesis of aldehyde 34.

Synthesis of Diastereomer 29
The synthesis of diastereomer 29 requires alteration of olefin geometry at C2-C3 . As shown in Scheme 6, a carbocupration of alkynyl ester 21 with Gilman reagent at 0 • C in the presence of TMSCl resulted in the desired E-olefin as a major isomer (E:Z = 7:3) [38]. DIBAL-H reduction furnished the alcohol 32, which was separated from its Z-isomer by flash column chromatography over silica gel. Protection of alcohol with tert-butyldimethylsilyl chloride and oxidative cleavage of the terminal olefin provided the C1-C6 fragment aldehyde 33 for diastereomer 29. Treatment of sulfone 5 with slightly less than one equivalent of KHMDS, followed by exposure to aldehyde 33, afforded the E-olefin. However, we found that the use of more than one equivalent base led to the epimerization of the chiral center on C5. Removal of the benzyl group followed by Dess-Martin oxidation [39,40] provided the C1-C15 fragment, aldehyde 34. ppm and 47.9 ppm, respectively, for the natural product). In addition, there is a distinction of chemical shifts at C24 (1.96 and 20.8 ppm, respectively, for synthetic 1 compared to 2.12 and 23.8 ppm, respectively, for natural 1). These discrepancies reveal that the α,βunsaturated double bond configuration might be E instead of Z. In addition, the structure may be an epimer at the C4 and C5 positions. Based on the NMR analysis, three diastereomers, 29, 30 and 31 (Figure 3), were designed. The syntheses of these structural variants were carried out utilizing our convergent synthetic route.

Synthesis of Diastereomer 29
The synthesis of diastereomer 29 requires alteration of olefin geometry at C2-C3. As shown in Scheme 6, a carbocupration of alkynyl ester 21 with Gilman reagent at 0 °C in the presence of TMSCl resulted in the desired E-olefin as a major isomer (E:Z = 7:3) [38]. DIBAL-H reduction furnished the alcohol 32, which was separated from its Z-isomer by flash column chromatography over silica gel. Protection of alcohol with tertbutyldimethylsilyl chloride and oxidative cleavage of the terminal olefin provided the C1-C6 fragment aldehyde 33 for diastereomer 29. Treatment of sulfone 5 with slightly less than one equivalent of KHMDS, followed by exposure to aldehyde 33, afforded the Eolefin. However, we found that the use of more than one equivalent base led to the epimerization of the chiral center on C5. Removal of the benzyl group followed by Dess-Martin oxidation [39,40] provided the C1-C15 fragment, aldehyde 34. The synthesis of diastereomer 29 is shown in Scheme 7. Brown asymmetric crotyllation, by utilizing (+)-B-methoxydiisopino-campheylborane and cis-2-butene with aldehyde 35 derived from 16, provided syn-alcohol 36 in good diastereoselectivity (8:1 dr). PMB protection and hydroboration-oxidation provided alcohol 37. Alcohol 37 was converted to sulfone 38, as described previously. A Julia-Kocienski olefination [24,25] between aldehyde 34 and sulfone 38 using KHMDS in 1,2-dimethoxyethane furnished (15E)-olefin in good selectivity (E:Z = 9:1). The use of THF as a solvent caused an increase in Z-olefin by-product (E:Z = 4:1). Removal of the PMB ether and primary TBS ether led to diol 39. MnO 2 oxidation and Pinnick oxidation [41] gave the corresponding seco-acid. Yamaguchi esterification furnished the macrolactone 40. A protecting group exchange protocol [26] afforded the corresponding vicinal diol. A one-pot reaction with the addition of HF•Py and Parikh-Doering oxidation led to isomer 29 exclusively, without isomerization of the exomethylene group at C11.
(15E)-olefin in good selectivity (E:Z = 9:1). The use of THF as a solvent caused an increase in Z-olefin by-product (E:Z = 4:1). Removal of the PMB ether and primary TBS ether led to diol 39. MnO2 oxidation and Pinnick oxidation [41] gave the corresponding seco-acid. Yamaguchi esterification furnished the macrolactone 40. A protecting group exchange protocol [26] afforded the corresponding vicinal diol. A one-pot reaction with the addition of HF•Py and Parikh-Doering oxidation led to isomer 29 exclusively, without isomerization of the exomethylene group at C11. Scheme 7. Synthesis of diastereomer 29.

Synthesis of Diastereomer 30
The synthesis of diastereomer 30 requires alteration of the stereochemistry at C9. Our synthesis started with commercially available (R)-D-malic acid. As shown in Scheme 8, reduction of malic acid with the borane dimethylsulfide complex furnished the corresponding triol. Selective protection of the diol with acetone and p-TsOH furnished the alcohol 41 with desired chirality at C9. Using the previous synthetic route, enantiomeric sulfone ent-8 was obtained. Sulfone ent-8 was then converted to the corresponding macrolactone, as described previously. Protecting the group exchange protocol and oxidation, followed by the removal of the TES group as described previously, provided diastereomeric structure 30, where the hydroxyl ketone stays as a δhydroxyl-ketone instead of cyclic hemiketal, as revealed from the analysis of its 1 H NMR and 13 C NMR spectral data.

Synthesis of Diastereomer 30
The synthesis of diastereomer 30 requires alteration of the stereochemistry at C9. Our synthesis started with commercially available (R)-D-malic acid. As shown in Scheme 8, reduction of malic acid with the borane dimethylsulfide complex furnished the corresponding triol. Selective protection of the diol with acetone and p-TsOH furnished the alcohol 41 with desired chirality at C9. Using the previous synthetic route, enantiomeric sulfone ent-8 was obtained. Sulfone ent-8 was then converted to the corresponding macrolactone, as described previously. Protecting the group exchange protocol and oxidation, followed by the removal of the TES group as described previously, provided diastereomeric structure 30, where the hydroxyl ketone stays as a δ-hydroxyl-ketone instead of cyclic hemiketal, as revealed from the analysis of its 1 H NMR and 13 C NMR spectral data.

Synthesis of Diastereomer 31
Our synthesis of this diastereomeric structure of iriomoteolide-1a required altering the configurations at the C 4 and C 5 chiral centers. Therefore, the enantiomeric C1-C5 segment aldehyde, ent-6, was synthesized as shown in Scheme 9. Our enzymatic resolution of racemic alcohol 19 provided nearly a 1:1 mixture of (R)-19 alcohol and its acetate derivative 42 in excellent yield. Saponification of acetate by treatment of K 2 CO 3 in MeOH at −30 • C provided (S)-19 alcohol in 92% ee. Seebach-Fráter alkylation [36] of (S)-19 with methyl iodide, as described in Scheme 3, furnished the corresponding anti-alcohol. Protection of alcohol as an MOM ether, followed by the reduction of the ester using LAH, afforded ent-20 alcohol. This was then converted to C1-C6 segment aldehyde ent-6. Aldehyde ent-6 was then exposed to Julia-Kocienski olefination with sulfone 5 to provide the corresponding trans-olefin, which was converted to the corresponding diastereomeric aldehyde, as described in Scheme 4. A second Julia-Kocienski olefination [24,25] with sulfone 4 provided the carbon framework for diastereomer 31. This was converted to the macrolactone, followed by the final target diastereomer 31 by following the steps described in Scheme 5.

Synthesis of Diastereomer 31
Our synthesis of this diastereomeric structure of iriomoteolide-1a required altering the configurations at the C4 and C5 chiral centers. Therefore, the enantiomeric C1-C5 segment aldehyde, ent-6, was synthesized as shown in Scheme 9. Our enzymatic resolution of racemic alcohol 19 provided nearly a 1:1 mixture of (R)-19 alcohol and its acetate derivative 42 in excellent yield. Saponification of acetate by treatment of K2CO3 in MeOH at −30 °C provided (S)-19 alcohol in 92% ee. Seebach-Fráter alkylation [36] of (S)-19 with methyl iodide, as described in Scheme 3, furnished the corresponding anti-alcohol. Protection of alcohol as an MOM ether, followed by the reduction of the ester using LAH, afforded ent-20 alcohol. This was then converted to C1-C6 segment aldehyde ent-6. Aldehyde ent-6 was then exposed to Julia-Kocienski olefination with sulfone 5 to provide the corresponding trans-olefin, which was converted to the corresponding diastereomeric aldehyde, as described in Scheme 4. A second Julia-Kocienski olefination [24,25] with sulfone 4 provided the carbon framework for diastereomer 31. This was converted to the macrolactone, followed by the final target diastereomer 31 by following the steps described in Scheme 5.

Synthesis of Diastereomer 31
Our synthesis of this diastereomeric structure of iriomoteolide-1a required altering the configurations at the C4 and C5 chiral centers. Therefore, the enantiomeric C1-C5 segment aldehyde, ent-6, was synthesized as shown in Scheme 9. Our enzymatic resolution of racemic alcohol 19 provided nearly a 1:1 mixture of (R)-19 alcohol and its acetate derivative 42 in excellent yield. Saponification of acetate by treatment of K2CO3 in MeOH at −30 °C provided (S)-19 alcohol in 92% ee. Seebach-Fráter alkylation [36] of (S)-19 with methyl iodide, as described in Scheme 3, furnished the corresponding anti-alcohol. Protection of alcohol as an MOM ether, followed by the reduction of the ester using LAH, afforded ent-20 alcohol. This was then converted to C1-C6 segment aldehyde ent-6. Aldehyde ent-6 was then exposed to Julia-Kocienski olefination with sulfone 5 to provide the corresponding trans-olefin, which was converted to the corresponding diastereomeric aldehyde, as described in Scheme 4. A second Julia-Kocienski olefination [24,25] with sulfone 4 provided the carbon framework for diastereomer 31. This was converted to the macrolactone, followed by the final target diastereomer 31 by following the steps described in Scheme 5. NMR spectra analysis of these diastereomers revealed that some individual chemical shifts, such as H4 and H24 of 29, H19 and H26 of 31, came closer to those of the natural product. These observations suggest that there may be an E-enoate and/or C4 and C5- NMR spectra analysis of these diastereomers revealed that some individual chemical shifts, such as H4 and H24 of 29, H19 and H26 of 31, came closer to those of the natural product. These observations suggest that there may be an E-enoate and/or C4 and C5epimers in the natural product. However, none of these isomers match the natural product. We carried out biological evaluations of the synthetic iriomoteolide-1a (1), -1b (2) and structural variants 29, 30, and 31. However, none of these compounds show any appreciable cytotoxicity. Yang and Dai's research groups also reported their independent synthetic approach of several other diastereomers, such as 43 [20,22], 44 [20], 45 [20], 46 [22] and 47 [20], as shown in Figure 4. Unfortunately, none of these structures match that of natural iriomoteolide-1a. The real structure of this biologically potent natural product remains veiled, waiting for collective effort in the synthetic community.
product. We carried out biological evaluations of the synthetic iriomoteolide-1a (1), -1b (2) and structural variants 29, 30, and 31. However, none of these compounds show any appreciable cytotoxicity. Yang and Dai's research groups also reported their independent synthetic approach of several other diastereomers, such as 43 [20,22], 44 [20], 45 [20], 46 [22] and 47 [20], as shown in Figure 4. Unfortunately, none of these structures match that of natural iriomoteolide-1a. The real structure of this biologically potent natural product remains veiled, waiting for collective effort in the synthetic community.

Materials and Methods
With regard to the general techniques used in this study, all moisture sensitive reactions were carried out under argon atmosphere. Anhydrous solvents were obtained as follows: THF and DME distilled from sodium and benzophenone; dichloromethane, toluene, triethylamine and diisopropylamine, distilled from CaH2. Column chromatography was performed with 230-400 mesh silica gel under low pressure of 5-10 psi. TLC was carried out with silica gel 60-F-254 plates, visualized under UV light and stained with phosphomolybdic acid. In addition, 1H NMR and 13C NMR spectra were recorded on Bruker Avance ARX-400 (400 and 100 MHz), or Bruker DRX500 (500 and 125 MHz) spectrometers. High and low resolution mass spectra were carried out by the Mass Spectroscopy Center at Purdue University. HPLC analysis and preparative HPLC were performed on Agilent 1100 Series instruments (Agilent Technologies, Santa Clara, CA, USA, Agilent 1200 Series Autosampler used for analytical work).

Materials and Methods
With regard to the general techniques used in this study, all moisture sensitive reactions were carried out under argon atmosphere. Anhydrous solvents were obtained as follows: THF and DME distilled from sodium and benzophenone; dichloromethane, toluene, triethylamine and diisopropylamine, distilled from CaH 2 . Column chromatography was performed with 230-400 mesh silica gel under low pressure of 5-10 psi. TLC was carried out with silica gel 60-F-254 plates, visualized under UV light and stained with phosphomolybdic acid. In addition, 1H NMR and 13C NMR spectra were recorded on Bruker Avance ARX-400 (400 and 100 MHz), or Bruker DRX500 (500 and 125 MHz) spectrometers. High and low resolution mass spectra were carried out by the Mass Spectroscopy Center at Purdue University. HPLC analysis and preparative HPLC were performed on Agilent 1100 Series instruments (Agilent Technologies, Santa Clara, CA, USA, Agilent 1200 Series Autosampler used for analytical work).

R)-5-(3-(tert-butyldimethylsilyloxy)-5-methylhex-5-enylthio)-1-phenyl-1H-tetrazole (8):
To a stirred solution of epoxide 14 (1.49 g, 6 mmol) and CuCN (54 mg, 0.6 mmol) in THF (40 mL) at −78 • C, we added isopropenylmagnesium bromide (3.6 mL, 0.9 mmol). The resulting suspension was warmed up to 0 • C and stirred for 30 min. The reaction mixture was cooled again to −78 • C and more vinylmagnesium bromide (12 mL, 6 mmol) was added dropwise. The reaction mixture was warmed up to 0 • C and stirred for 1 h, before 20 mL of saturated NH 4 Cl and 10 mL of NH 4 OH were added to quench the reaction. The layers were separated and the aqueous layer was extracted with diethyl ether (3 × 30 mL). The combined organic extracts were washed with brine and dried over anhydrous magnesium sulfate. Filtration and concentration under reduced pressure gave a crude product. Flash chromatography on silica gel (20% EtOAc/hexanes) afforded the corresponding alcohol as a colorless oil (1.6 g, 92%). To a stirred solution of the above alcohol (942mg, 3.24 mmol) in DMF (6 mL), we added imidazole (353 mg, 5.18 mmol) and TBSCl (538 mg, 3.57 mmol), respectively, at 0 • C. The reaction mixture was stirred at 23 • C for 12 h. A solution of saturated NaHCO 3 (aq) was added and the aqueous layer was extracted by diethyl ether . The combined organic extracts were washed with water and brine, dried over anhydrous MgSO 4 , filtered, and concentrated in vacuo. Flash chromatography (4% EtOAc/hexanes) gave the silyl To a stirred solution of diisopropylamine (15.8 mL, 0.112 mol) in THF (80 mL) at −78 • C, we added n-BuLi (1.6 M in hexane, 71.8 mL, 0.107 mol) dropwise. The mixture was kept at −78 • C for 20 min before a solution of the above alcohol (7.4 g, 42.9 mmol) was added dropwise. After another 20 min of stirring, MeI (6.7 mL, 0.107 mol) was added to the reaction mixture in a dropwise manner. The reaction was stirred at −10 • C for 4 h before 50 mL sat NH 4 Cl was added. The organic layer was separated and the aq layer was extracted with diethyl ether (3 × 50 mL). The combined organic solution was washed with water and brine, dried over anhydrous MgSO 4 , filtered, and concentrated in vacuo. Flash chromatography on silica gel (10% EtOAc/hexanes) afforded the anti-methyl alcohol  6 mL, 0.105 mol) was added slowly. The reaction mixture was stirred at −78 • C for 1 h and allowed to warm up to room temperature and stirred for 30 min, before pouring it into 1M NaHSO 4 solution. The organic layer was separated and the aqueous layer was extracted with DCM. The combined organic extracts were washed with brine, dried over anhydrous Na 2 SO 4 , filtered, and concentrated in vacuo to give crude aldehyde (3.35 g), which was used in the next step without further purification. R f value (EtOAc/hexane 1:2) 0.7.
To a stirred solution of the CBr 4 (13.86 g, 41.8 mmol) in DCM (50 mL) at 0 • C, we added PPh 3 (21.93 g, 83.6 mmol). The reaction mixture was stirred at 0 • C for 10 min. A solution of the above aldehyde in DCM (10 mL) was added dropwise. The mixture was warmed up to 23 • C and stirred for 30 min. The reaction was poured into saturated NaHCO 3 (aq). The aqueous layer was extracted by DCM. The combined organic extracts were washed with brine and dried over anhydrous Na 2  To a stirred solution of the above dibromide (3.88 g, 10.1 mmol) in THF (30 mL), we added n-BuLi (1.6 M in hexanes, 19.0 mL, 30.3 mmol) at −78 • C. The mixture was stirred at −78 • C for 15 min before methyl chloroformate (2.34 mL, 30.3 mmol) was added. The reaction mixture was stirred at −78 • C for 1 h before pouring it into saturate NH 4 Cl (aq). The organic layer was separated and the aqueous layer was extracted by Et 2 O (3 × 50 mL). The organic extracts were combined, washed by water and brine, dried over anhydrous MgSO 4 , filtered, and concentrated in vacuo. Flash chromatography on silica gel (5% EtOAc/hexanes) produced alkynyl ester 21 (2.14 g, 99%) as a pale yellow oil. To a stirred solution of the above alcohol (1.45 g, 7.24 mmol) in DCM (50 mL), we added imidazole (739 mg, 10.9 mmol) and TBSCl (1.2 g, 7.96 mmol) at 0 • C. The reaction was warmed up to rt and stirred for 1 h, before pouring it into a mixture of sat NaHCO 3 (50 mL) and crushed ice. The mixture was extracted with ethers (3 × 60 mL) and the organic layer was washed with water and brine, dried over anhyd MgSO 4 and evaporated in vacuo to give the crude TBS ether as a clear oil, which was used for the next step without further purification. Flash chromatography on silica gel (25% EtOAc/hexanes) afforded the silyl ether (2.28 g, 99%). To a stirred solution of above olefin (1.09 g, 3.47 mmol) in dioxane (24 mL) and water (8 mL), we added 2,6-lutidine (2.02 mL, 17.4 mmol), OsO 4 (2.5% in t-BuOH, 1.74 mL, 0.14 mmol) and NaIO 4 (2.97 g, 12.9 mmol) at 0 • C. The mixture was stirred at 0 • C for 18 h before saturated NaHCO 3 (10 mL) and NaS 2 O 3 (10 mL) were added. The mixture was stirred for another 30 min, extracted by EtOAc. The combined organic extracts were dried over anhydrous Na 2 SO 4 , filtered, and concentrated in vacuo. Flash chromatography on silica gel (25% EtOAc/hexanes) produced the aldehyde 6 (736 mg, 67%).

Aldehyde (3):
To a stirred solution of benzyl ether 23 (550 mg, 0.72 mmol) in THF (10 mL) and allyl ethyl ether (1 mL), we transferred a soln of lithium metal (50 mg, 7.2 mmol) in liquid ammonia (12 mL) in portions at −78 • C. The reaction was carefully monitored by TLC and stopped immediately after the solution became slightly blue. Ammonium chloride (2 g) was added to quench the reaction. The mixture was allowed to warm up to rt to evaporate the ammonia, before water (10 mL) was added. The organic layer was separated and the aq layer was extracted with diethyl ether (3 × 20 mL). The combined organic extracts were washed with water and brine, dried over anhyd MgSO 4 and evaporated in vacuo. The crude product was purified by flash chromatography (EtOAc/hexane 1:10) to give the alcohol as a colorless oil (402 mg, 83% yield), along with the recovered starting material.  To a suspension of the above alcohol (352 mg, 0.53 mmol) and sodium bicarbonate (265 mg, 3.2 mmol) in DCM (20 mL), we added Dess-Martin periodinane (445 mg, 1.05 mmol) at rt. The reaction mixture was stirred for 1 h before it was poured into a mixture of sat NaHCO 3 (10 mL) and sodium thiosulfate (10 mL). The organic layer was separated and the aq layer was extracted with diethyl ether (3 × 20 mL). The combined organic extracts were washed with water and brine, dried on anhyd MgSO 4 and evaporated in vacuo to give the crude aldehyde 3 (352 mg, quantitative), which was used directly in the next step without further purification. 1  To a stirred solution of alcohol 12 (7.31 g, 73 mmol) in DMF (70 mL), we added imidazole (5.96 g, 87.6 mmol) and TBSCl (11 g, 73 mmol) at 0 • C. The reaction was warmed up to rt and stirred for 6 h. The reaction mixture was then poured into a mixture of sat NaHCO 3 (50 mL) and crushed ice. The mixture was extracted with ethers (3 × 60 mL) and the organic layer was washed with water and brine, dried over anhyd MgSO 4 and evaporated in vacuo to give the crude TBS ether as a clear oil, which was used for the next step without further purification. To a stirred solution of thus obtained olefin in THF (60 mL), we added the BH 3 ·THF complex (73 mL, 1 M soln in THF, 73 mmol) at 0 • C. The reaction mixture was allowed to warm up to rt and stirred for 6 h. NaOH (10 mL) and H 2 O 2 (15 mL, 70% soln) were added and the mixture was refluxed for 1 h. The organic layer was separated and the aq layer was extracted with Et 2 O; the combined organic layer was washed with water and brine, dried over MgSO 4  1 mmol) in DCM (10 mL) was transferred in at the same temperature. The reaction mixture was stirred for 30 min before Et 3 N (10.5 mL, 75.5 mmol) was added. After stirring at −78 • C for 1 h, the reaction mixture was allowed to warm up to 0 • C for 30 min, before it was poured into sat NaHCO 3 soln (30 mL). The organic layer was separated and the aq layer was extracted with diethyl ether (3 × 20 mL). The combined organic extracts were washed with water and brine, dried over anhyd MgSO 4 and evaporated in vacuo. The crude product was purified by flash chromatography (EtOAc/hexane 1:30) to give the aldehyde as a colorless oil (3.03 g, 87% yield), which was used in the next step immediately. R f value (EtOAc/hexane 1:10): 0.85.
To a stirred mixture of potassium tert-butoxide (8.7 mL, 1.0 M soln in THF, 8.7 mmol) and trans-2-butene (1.4 mL, 14.5 mmol) in THF (30 mL), we added n-butyllithium (5.5 mL, 1.6 M soln in THF, 8.7 mmol) at −78 • C. After complete addition of n-butyllithium, the mixture was stirred at −45 • C for 10 min. The resulting orange solution was cooled to −78 • C again and a solution of (−)-Ipc 2 BOMe (3.3 g, 10.4 mmol) in THF (10 mL) was added dropwise. After 30 min of stirring, boron trifluoride etherate (1.5 mL, 11.6 mmol) was added dropwise. Then, the above aldehyde (1.34 g, 5.8 mmol) in THF (5 mL) was transferred in. The mixture was stirred at −78 • C for 3 h before NaOH (6.8 mL, 3 M soln) and H 2 O 2 (4.7 mL, 70% soln) were added. The contents were refluxed for 1 h. The organic layer was separated and the aq layer was extracted with diethyl ether (3 × 30 mL). The combined organic extracts were washed with water and brine, dried over anhyd MgSO 4  we added NaH (60%, 150 mg, 3.7 mmol) at 0 • C. The mixture was stirred for 30 min before PMBCl (0.5 mL, 3.7 mmol) was added at 0 • C. After stirring at rt overnight, water (4 mL) and Et 2 NH (2 mL) were added and the mixture was stirred for 1h, before it was poured into sat NaHCO 3 (aq). The mixture was extracted with diethyl ether (3 × 30 mL). The combined organic extracts were washed with water and brine, dried over anhyd To a stirred solution of the above olefin (1.45 g, 3.6 mmol) in THF (30 mL), we added 9-BBN (14.2 mL, 0.5 M soln in THF, 7.2 mmol) at 0 • C. The reaction mixture was allowed to warm up to rt and stirred for 3 h. NaOH (0.6 mL) and H 2 O 2 (4 mL) were added and the mixture was refluxed for 1 h. The organic layer was separated and the aq layer was extracted with Et 2 O; the combined organic layer was washed with water and brine, dried over Na 2  we added a solution of ammonium molybdate (320 mg, 0.26 mmol) in hydrogen peroxide (1.6 mL) and water (0.8 mL) at rt. The reaction mixture was stirred for 12 h and poured into a mixture of sat NaHCO 3 (10 mL) and sodium thiosulfate (10 mL). The organic layer was separated and the aq layer was extracted with diethyl ether (3 × 20 mL). The combined organic extracts were washed with water and brine, dried on anhyd MgSO 4 and evaporated in vacuo. The crude product was purified by flash chromatography (EtOAc/hexane 1:12) to give the sulfone 4 as a colorless oil (352 mg, 95% yield    8, 39.7, 39.2, 37.5, 35.7, 35.6, 32.8, 29.7, 28.5, 26.6, 26, 25.9, 20.6, 20.4, 18.7, 18.4, 18, 15.5, 15.4, 15, 6 mL), we added DDQ (88 mg, 0.4 mmol) at 0 • C and the reaction mixture was stirred at that temperature for 1 h. The reaction was quenched by sat NaHCO 3 (5 mL) and the organic layer was separated. The aq layer was extracted with diethyl ether (3 × 20 mL). The combined organic extracts were washed with sat NaHCO 3 , water and brine, dried on anhyd MgSO 4 and evaporated in vacuo. The crude product was purified by flash chromatography (EtOAc/hexane 1:15) to give the corresponding alcohol as a colorless oil (138 mg, 76% yield).  13  To a stirred solution of the above TBS ether (115 mg, 0.12 mmol) in methanol (4 mL), we added ammonium fluoride (125 mg, 3.37 mmol) at rt and the reaction mixture was stirred at that temperature for 8 h. Et 2 O (30 mL) was added to precipitate the ammonium fluoride, which was removed by suction filtration. The crude product was purified by flash chromatography (EtOAc/hexane 1:4) to give the alcohol 25 as a colorless oil (94 mg, 95% yield). To a stirred solution of the above aldehyde (105 mg, 0.12 mmol) and 2-methyl-2-butene (2 mL) in tert-butanol (8 mL), we added a solution of NaH 2 PO 4 . H 2 O (200 mg) and NaClO 2 (200 mg), dropwise, in H 2 O (2 mL) at 0 • C. In addition, the reaction mixture was allowed to warm up to rt and stirred for 30 min. The reaction was poured into water (5 mL) and extracted with ethyl acetate (3 × 20 mL). The combined organic extracts were washed with water and brine, dried over anhyd Na 2 SO 4 and evaporated in vacuo. The crude product was purified by flash chromatography (MeOH/chloroform 3:100) to give the seco-acid as a colorless oil (97 mg, 89% yield for two steps). R f value (EtOAc/hexane 1:2): 0.68.
To the solution of thus obtained seco-acid in THF (4 mL), we added DIPEA (0.33 mL, 1.91 mmol) and 2,4,6-trichlorobenzoyl chloride (0.2 mL, 1.27 mmol) at rt. The reaction was stirred for 3 h at that temperature, before the THF solvent was removed by vacuo. The the residue toluene (10 mL) was added and the solution was transferred to a stirred solution of DMAP (388 mg, 3.18 mmol) in toluene (150 mL) at rt over 16 h, through a syringe pump. The resulting mixture was stirred at rt for 36 h and poured into sat NaHCO 3 (20 mL). The organic layer was separated and the aq was extracted with diethyl ether (3 × 20 mL). The combined organic phase was washed with water and brine, dried over anhyd  Macrolactone alcohol (27): To a stirred solution of macrolactone 26 (52 mg, 0.063 mmol) in THF (4 mL), we added pyridine (1 mL) followed by HF . pyridine complex (70%,