Total Synthesis and Structural Reassignment of Laingolide A

The asymmetric total synthesis of four diastereomers of laingolide A was achieved, which led to the unambiguous assignment of the stereochemistry of the natural product. The salient features of the convergent, fully stereocontrolled approach were a copper-catalysed stereospecific Kumada-type coupling, a Julia-Kocienski olefination and an RCM/alkene migration sequence to access the desired macrocyclic enamide.


Introduction
The modern structure determination of unknown natural products remains a challenging problem, especially when a small quantity of the natural compound is available, limiting the full possibility of the modern spectroscopic methods. For this reason, total synthesis has played a major role in the structure elucidation and revision of complex natural products for a long time. In the absence of a firm structural assignment, a combination of the stereochemical logic of the synthesis and spectroscopic comparison could be employed as tools to establish the correct structure of natural products [1][2][3][4]. Previous work in our group led to the reassignment of the configuration of a number of marine natural products [5][6][7][8][9][10][11]. These results encouraged us to embark upon the synthesis of other natural products with unknown configurations. We describe herein the determination of the complete relative and absolute stereochemistries of (+)-laingolide A and the total synthesis of this material.
Laingolide A (1), along with madangolide (2), was isolated in 1999 from the marine cyanobacterium Lyngbya bouillonii collected in Papua New Guinea [12]. The very same blue-green algae also produced laingolide (3) (Scheme 1), which was disclosed as the first member of the novel macrolide family [13]. Additionally, another chlorinated analogue, laingolide B (4), was isolated in 2010 by Luesch and co-workers from the same species of bacteria collected in Apra Harbor, Guam [14]. The planar structures of the laingolides were established using a combination of detailed 1D and 2D NMR analysis. However, these macrolides underwent degradation over time, which hampered progress towards complete assignments of their absolute stereochemistry. In 2010, Gerwick and co-workers reported the isolation of palmyrolide A (5), a structurally closely related 15-member macrolide from a cyanobacterial assemblage comprised of Leptolyngbya and Oscillatoria species collected at Palmyra Atoll, south of Hawaii [6]. One of the absolute configurations present in palmyrolide A was correctly assigned upon its initial isolation [15], and the absolute configurations of the remaining stereocentres were later established via total syntheses [16][17][18][19][20][21][22][23][24]. The structurally intriguing laingolides have attracted considerable attention from the synthetic community [25][26][27]. In 2018, Dai and co-workers reported the first total synthesis of laingolide B and unambiguously assigned the absolute configuration as depicted in structure 4 [27]. Laingolide A (1) was isolated from the bacteria collected in a different location and eleven years before the isolation of laingolide B (4). Laingolide A contains It would be interesting to find out whether the absolute configurations of laingolide A (1) were likely the same based on the possible similar biogenesis to palmyrolide A (5). Through the completion of the synthesis of four diastereomers, we shall be able to provide conclusive evidence for the absolute and relative stereochemistry of laingolide A (1) [28].
Structurally, laingolide A features a 15-member macrocyclic core, which is composed of a sterically encumbered ester derived from a tert-butyl carbinol, a trans-N-methyl enamide subunit and two chiral methyl appendages. At the outset of this synthetic venture, our primary objective was to rapidly access four diastereomers (Scheme 2) and thus conclusively establish the absolute configuration of laingolide A. With this in mind, we opted for a modular and flexible approach as we pondered its retrosynthetic analysis (Supplementary Materials) (Scheme 2) [29][30][31][32][33][34][35][36]. We envisioned that the four diastereomers of laingolide A could be constructed from fragments 6, 7, 8 and ent-8 via three key transformations, as illustrated in Scheme 2. This highly convergent strategy relied on a ring-closing metathesis (RCM) at C-12 and C-13 to deliver the macrocycle and olefin migration to forge the enamide moiety at the final stage [19,22,27]. Both copper-catalysed Kumada-type coupling [37] of cyclic sulfate esters and Julia-Kocienski olefination [38][39][40] were then employed to construct the RCM precursor. Scheme 2. Retrosynthetic analysis of laingolide A and its diastereomers (1a-d).

Results
The synthesis towards the chiral aldehydes (Scheme 3) commenced with the known chiral 1,3-hydroxy ketone 9 [23], which was prepared via List's proline-catalysed aldolisation between acetone and pivalaldehyde [41]. A three-step sequence [23] was employed to elaborate 1,3-hydroxy ketone 9 into cyclic sulfate ester 6 involving (1) the syn reduction of ketone 9 with DIBAL-H, (2) conversion of the syn-diol into the corresponding sulfite with thionyl chloride and (3) oxidation of the cyclic sulfite with NaIO 4 in the presence of catalytic amounts of RuCl 3 . Nucleophilic ring-opening of the cyclic sulfate 6 using a mixed organometallic reagent derived from allylmagnesium chloride and stoichiometric quantities of copper(I) iodide has already been reported [42]. Recently, a catalytic version of this reaction, also termed C(sp 3 )-C(sp 3 ) Kumada-type coupling of cyclic sulfate esters was reported [37]. We opted to incorporate this catalytic reaction into our synthesis. Thus, treatment of cyclic sulfate 6 and 10 mol% of cuprous iodide in THF with allyl magnesium bromide at −20 • C followed by hydrolysis of the corresponding intermediate gave rise to the corresponding alcohol with a 75% yield with a >95:5 diastereomeric ratio (dr), as determined using 1 H NMR spectroscopy. This reaction occurred at the least-hindered site, with the complete inversion of the configuration at that centre. Protection of the resulting alcohol with TBSOTf (tert-butyldimethylsilyl trifluoromethanesulfonate) and triethylamine afforded TBS ether 10 with a 96% yield. Hydroboration of 10 with 9-borabicyclo[3.3.1] nonane (9-BBN) and oxidation of the resulting organoborane (NaHCO 3 , H 2 O 2 ) furnished alcohol 11 with an 89% yield, which in turn was subjected to oxidation with TEMPO, NaOCl and NaBr [43] to provide aldehyde 12 with an 85% yield. In parallel, the hydroxyl-directed antireduction of hydroxy ketone 9 with the Evans-Carreira protocol [44] proceeded smoothly to furnish the desired 1,3-anti diol with a diastereomeric ratio of 5:1 (determined by 1 H NMR of the crude reaction mixture). These diastereomers were separated using flash chromatography and the major one was used in subsequent reactions. The anti-substituted cyclic sulfate 7 was prepared with a 60% yield over three steps using the same procedure as described for 6. The elaboration of the substituted cyclic sulfate 7 into aldehyde 15 was accomplished in a way similar to that described for the preparation of aldehyde 12. With a reliable route to useful quantities of the required aldehydes 12 and 15 in hand, our efforts turned to the divergent total synthesis of laingolide A (Scheme 4). This required the combination of aldehydes 12 and 15 separately with BT-sulfones 8 and then with ent-8 [45] (Scheme 4a). Under the optimum conditions investigated, each aldehyde (12 or 15) underwent condensation with 1.2 molar equivalent of sulfone (8 or ent-8) treated with 1.2 molar equivalent of NaHMDS in toluene at −78 • C to afford the corresponding alkene as a mixture of geometrical isomers (Z:E ≈ 3-7:1) in high yield. Next, each of the resultant internal alkenes (16,20,24,28) was separately subjected to palladium-chloridemediated hydrogenation in ethanol with the concomitant removal of the TBS ethers that furnish the corresponding diol (17,21,25,29) [46]. The primary alcohol of the above diol was selectively oxidised with TEMPO in the presence of bis-acetoxyiodobenzene (BAIB) [47] and the resulting carboxylic acid was then coupled with the N-methylallylamine by utilising EDCI-HOAt and DMAP as a base to provide the corresponding amide (18,22,26,30). For the conversion to the required diene (19,23,27,31), each amide alcohol was separately acylated with acryloyl chloride in the presence of triethylamine and DMAP. The four diastereomeric dienes (19,23,27,31) were separately subjected to ring-closing metathesis using the second-generation Grubbs catalyst (G-II) to afford the corresponding unsaturated macrolactone as isomeric mixtures, which were subsequently treated with RuH(PPh 3 ) 3 (CO)Cl in refluxing toluene [48] to furnish the desired enamides 1a-d with good yields. The comparison of the spectral data of 1a-d with the reported spectra of laingolide A was informative. Compound 1a, featuring a C(7)-R methyl beta to the C(9)-R-tert-butyl-substituted stereocenter, the same as that of natural palmyrolide A (5), did not match the literature values reported for the laingolide A. This suggested that the biogenesis of the laingolide A and palmyrolide A might follow different pathways or that C7 is epimerized at some stage of the biosynthesis of laingolide A (or of palmyrolide A). It was clear from comparing the 13 C NMR data (Scheme 4b) that diastereomer 1c represented the correct structure of natural laingolide A. The absolute stereochemical assignment of laingolide A was thus assigned as 2S,7S,9R, as shown in 1c (Scheme 4).

General Information
All reactions were conducted in flame-dried or oven-dried glassware under an atmosphere of dry nitrogen or argon. Oxygen and/or moisture-sensitive solids and liquids were transferred appropriately. The concentration of solutions in vacuo was accomplished using a rotary evaporator fitted with a water aspirator. Residual solvents were removed under a high vacuum (0.1-0.2 mm Hg). All reaction solvents were purified before use: tetrahydrofuran (THF) was distilled from Na/benzophenone. Toluene was distilled over molten sodium metal. Dichloromethane (DCM), 1,2-dichloroethane (DCE) and trimethylamine (Et 3 N) were distilled from CaH 2 . Methanol (MeOH) was distilled from Mg/I 2 . The reagents were purchased at the highest commercial quality and used without further purification, unless otherwise stated. Flash column chromatography was performed using the indicated solvents on E. Qingdao silica gel 60 (230-400 mesh ASTM). Reactions were monitored using thin-layer chromatography (TLC), which was carried out using pre-coated sheets (Qingdao silica gel 60-F250, 0.2 mm, Tsingtao, China). Compounds were visualised with UV light, iodine and ceric ammonium molybdate stainor phosphomolybdic acid in EtOH. The 1 H NMR spectra were recorded on Bruker Avance 300 MHz, Avance 400 MHz or Avance 500 MHz spectrometers (Karlsruhe, Germany). Chemical shifts were reported in parts per million (ppm), relative to either a tetramethylsilane (TMS) internal standard or the signals due to the solvent (Supplementary Materials). The following abbreviations are used to describe the spin multiplicity: s = singlet, d = doublet, t = triplet, q = quartet, qn = quintet, m = multiplet, br = broad, dd = doublet of doublets, dt = doublet of triplets, dq = doublet of quartets, ddd = doublet of doublet of doublets; other combinations are derived from those listed above. Coupling constants (J) are reported in hertz (Hz) for corresponding solutions, and chemical shifts are reported as parts per million (ppm) relative to residual CHCl 3 δH (7.26 ppm). 13 C Nuclear magnetic resonance spectra were recorded using a 75 MHz, 100 MHz or 125 MHz spectrometer (Karlsruhe, Germany) for corresponding solutions, and chemical shifts are reported as parts per million (ppm) relative to residual CDCl 3 δC (77.16 ppm) (Supplementary Materials). High-resolution mass spectra were measured on an ABI Q-star Elite (Beijing, China). Optical rotations were recorded on a Rudolph AutoPol-I polarimeter (Shanghai, China) at 589 nm with a 50 mm cell. Data are reported as follows: specific rotation (c (g/100 mL), solvent).

Conclusions
In summary, we have unambiguously established the relative and absolute configuration of laingolide A through the total synthesis of four diastereomers of the natural product. The key features of the convergent and fully stereocontrolled route included a copper-catalysed stereospecific Kumada-type coupling, a Julia-Kocienski olefination and an RCM/alkene migration sequence to access the desired macrocyclic enamide.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/md19050247/s1: initial approaches via the cross-metathesis and 1 H-and 13 C-NMR charts of all the compounds.
Author Contributions: F.W., Y.G. and T.Y. conceived and designed this research; F.W. and T.Z. prepared the compounds and collected their spectral data; F.W., J.Y. and Y.G. analysed the experimental data; Y.G. and T.Y. prepared the manuscript. All authors have read and agreed to the published version of the manuscript.