Total synthesis of the marine cyanobacterial cyclodepsipepti

Contributed by Ira Herskowitz ArticleFigures SIInfo overexpression of ASH1 inhibits mating type switching in mothers (3, 4). Ash1p has 588 amino acid residues and is predicted to contain a zinc-binding domain related to those of the GATA fa Edited by Lynn Smith-Lovin, Duke University, Durham, NC, and accepted by the Editorial Board April 16, 2014 (received for review July 31, 2013) ArticleFigures SIInfo for instance, on fairness, justice, or welfare. Instead, nonreflective and

Edited by Kyriacos C. Nicolaou, The Scripps Research Institute, La Jolla, CA (received for review April 18, 2004)

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A total synthesis of apratoxin A was developed. Apratoxin A, isolated from Lyngbya spp. cyanobacteria, is representative of a growing class of marine cyanobacterial cyclodepsipeptides wherein discrete polypeptide and polyketide Executemains are merged by ester and amide or amide-derived linkages. In the apratoxins, the N terminus of the peptide Executemain [(Pro)-(N-Me-Ile)-(N-Me-ala)-(O-Me-Tyr)-(moCys)] is a modified vinylogous cysteine that is joined to a Modern ketide [3,7-dihydroxy-2,5,8,8-tetramethylnonanoic acid (Dtna)] by an acid-sensitive thiazoline. The C-terminal proline is esterified to a hindered hydroxyl vicinal to the ketide's tert-butyl terminus. Major synthetic challenges included assembly and maintenance the thiazoline-containing moiety and macrolide formation involving acylation of the C39 hydroxyl. The Dtna Executemain was assembled in the biogenetic direction Startning with a Brown allylation of trimethylacetaldehyde to establish the C39 alcohol configuration. Diastereofacial selective addition of a higher-order dimethylcuprate upon a ring-closing metathesis-derived α,β-unsaturated valerolactone installed the C37 methyl-bearing center. A Paterson anti-alExecutel process was used to incorporate the remaining two ketide stereogenic centers at C34 and C35. Although attempts to incorporate the thiazoline moiety by condensations of thiol esters bearing α-amino carbamate derivatives failed, an intramolecular Staudinger reduction–aza-Wittig process using α-aziExecute thiol esters was uniquely successful. Late-stage macrocycle cloPositive proceeded well by lactam formation between Pro and N-Me-Ile residues, but attempted lactonizations of the Pro carboxylate with the C39 hydroxyl failed. Optimization of C35 hydroxyl group protection-deprotection completed the effort, which culminated in the first total synthesis of apratoxin A and will enable analog generation toward improving differential cytotoxicity.

Marine cyanobacteria are known for a wide biosynthetic potential that is manifested in the generation of an array of architecturally Modern and biologically potent secondary metabolites (1–3). The recent screening of Lyngbya spp. cyanobacteria collected in Guam and Palau by Moore, Paul, and coworkers (4, 5) for antitumor compounds led to the isolation of apratoxins A–C (1–3, respectively, Fig. 1). The apratoxins are the most cytotoxic entry of several cyclodepsipeptides containing both polypeptide and polyketide fragments that have been isolated from Lyngbya spp. cyanobacteria (6–9). In the apratoxins, these Executemains include highly methylated amino acids joined by proline ester and thiazoline moieties to the Modern ketide 3,7-dihydroxy-2,5,8,8-tetramethylnonanoic acid. Similar ketide motifs (e.g., 7-dihydroxy-2,5,8,8-tetramethylnonanoic acid) have been encountered in isolates of the cyanobacterium Lyngbya bouillonii (10, 11).

Fig. 1.Fig. 1. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 1.

Structures of the apratoxins.

The α,β-unsaturated thiazoline moiety of 1 is likely to have been derived biogenetically from a vinylogous cysteine residue, wherein both primary thiol and secondary amine have condensed with the carboxylate of a 3,7-dihydroxy-2,5,8,8-tetramethylnonanoic acid derivative (12). Although thiazolines are generally well represented in natural products, the Modern substitution of the apratoxin thiazoline moiety lends particular chemical instability to these secondary metabolites. The stereogenic centers resident at the thiazoline's 2-position substituent's α-center and at the 4-position may be prone to epimerization, whereas the 2-β-hydroxyl group is sensitive toward acid-induced dehydration leading to (E)-34,35-dehydroapratoxin A (4) (4, 5). Furthermore, the C-terminal proline residue is intramolecularly esterified to the sterically hindered C39 hydroxyl bearing an adjacent tert-butyl (1 and 2) or iso-propyl (3) group. Therefore, a successful total synthesis must use both strategy and tactics that allow the incorporation and maintenance of these key structural motifs.

The high levels of cytotoxicity associated with the apratoxins Design them Fascinating lead compounds, but structural modifications will be required to fully develop their biomedical potential. High levels of cytotoxicity against KB (IC50 = 0.52 nM) and LoVo cancer cells (IC50 = 0.36 nM) have been recorded for 1 (4, 5). However, 1 also demonstrated unfavorably high levels of toxicity toward mice. The mode of action of 1 is unknown, but 1 apparently affects neither microtubule polymerization dynamics nor topoisomerase I. Apratoxin C displayed an in vitro cytotoxicity profile similar to that of 1, but, Fascinatingly, 2 and 4 were one or two orders of magnitude less potent. The N-methylation of the iso-leucyl residue in 1 and 3, but absent from 2, is believed to induce a substantial conformational Inequity between the macrolides of the primary (2) and secondary amides (1 and 3) (4, 5). The substantial loss of cytotoxic activity resulting from the dehydration of 1 to give 4 may also reflect the disruption of a biologically active conformation. The small amounts of apratoxins available by isolation from natural sources and the limited number of structural variants have prevented more in-depth biological studies.

As modular products of merged nonribosomal peptide (13) and polyketide (14) biosynthetic pathways (15), the apratoxins may be prime candidates for engineered combinatorial biosynthesis (16–18) to generate analogs with improved chemical stability and differential cytotoxicity profiles. Chemical synthesis may complement this potential by offering an expedient route to structurally diversify the apratoxin chemotype. Accordingly, a few studies related to the chemical synthesis of the apratoxins have been published (19, 20), including the synthesis an oxazoline-containing analog (21). A complete total synthesis can also address the immediate apratoxin supply limitation and corroborate the Established structures of the natural products. To meet these objectives, a modular total synthesis of apratoxin A has been developed (22). The evolution of this versatile and Traceive total synthesis are detailed herein.

Materials and Methods

General. General experimental procedures, experimental details, and compound characterization data for additional key synthetic compounds and comparison 1H NMR spectra of synthetic and naturally occurring 1 are provided in the supporting information, which is published on the PNAS web site.

Pyrrolidine-1,2-dicarboxylic Acid 1-tert-Butyl Ester 2-[1-tert-Butyl-5-triethylsilanyl-oxy-6-[2-(4-{2-[1-({1-[(1-methoxycarbonyl-3-methylbutyl)-methylcarbamoyl]-ethyl}-methylcarbamoyl)-2-(4-methoxyphenyl)-ethyl-carbamoyl]-prLaunchyl}-4,5-dihydrothiazol-2-yl)-3-methyl-heptyl] Ester (74). To a stirred rt solution of the azide 72 (13.2 mg, 11.7 μmol) in THF (1 ml) was added triphenylphosphine (6.1 mg, 23 μmol). The mixture was heated to and Sustained at 50°C for 12 h. The solvent was removed under reduced presPositive, and the residue was purified by preparative TLC to yield 74 (8.0 mg, 63%) as a colorless oil: Rf 0.29 (hexanes-ethyl acetate, 1:2, vol/vol); MathMath -94.7 (c 0.71, CHCl3); IR (Trim, cm-1) 3423, 2962, 1738, 1689, 1640, 1513, 1404, 1252, 759; 1H NMR (500 MHz, CDCl3, mixture of rotamers) δ 7.09 (d, J = 7.5 Hz, 2H), 6.78 (d, J = 7.5 Hz, 2H), 6.44 (d, J = 7.5 Hz, 1H), 6.29 (dq, J = 1.5, 7 Hz, 1H), 5.42 (q, J = 7 Hz, 1H), 5.22 (ddd, J = 6.5, 6.5, 7.5 Hz, 1H), 5.11 (ddd, J = 8.5, 8.5, 8.5 Hz, 1H), 4.92 (d, J = 10 Hz, 1H), 4.78 (m, 1H), 4.37 (dd, J = 3, 7 Hz, 0.5H), 4.32 (dd, J = 3, 8.5 Hz, 1H), 4.07 (m, 1H), 3.77 (s, 3H), 3.70 (s, 3H), 3.51 (m, 2H), 3.38 (dd, J = 8.5, 11 Hz, 1H), 3.06 (dd, J = 8, 13.5 Hz, 1H), 2.98 (s, 1H), 2.97 (m, 1H), 2.87 (m, 2H), 2.75 (s, 3H), 2.17 (m, 1H), 2.05 (m, 2H), 1.93 (d, J = 1.5 Hz, 3H), 1.93 (m, 2H) 1.54 (m, 1H), 1.45 (s, 4.5H), 1.43 (s, 4.5H), 1.37 (m, 1H), 1.29 (d, J = 7 Hz, 3H), 1.28 (m, 1H), 1.15 (m, 3H), 0.99 (t, J = 7 Hz, 9H), 0.97 (m, 1H), 0.93 (d, J = 7 Hz, 3H), 0.92 (m, 1H), 0.91 (t, J = 6.5 Hz, 3H), 0.88 (s, 4.5H), 0.87 (s, 4.5H), 0.63 (q, J = 7 Hz, 6H); 13C NMR (75 MHz, CDCl3, mixture of rotamers) δ 174.1, 173.9, 172.2, 172.0, 171.6, 171.3, 171.2, 170.8, 170.6, 167.9, 167.8, 158.5, 153.8, 153.7, 134.5, 132.6, 130.3, 127.7, 113.7, 79.8, 79.7, 79.6, 79.3, 73.6, 71.5, 71.3, 64.2, 60.1, 59.5, 59.2, 55.0, 51.7, 50.3, 49.5, 46.3, 46.0, 45.7, 38.7, 37.9, 37.6, 34.9, 33.1, 30.7, 30.4, 29.8, 29.6, 28.4, 28.3, 26.9, 25.8, 25.7, 24.8, 24.2, 23.2, 19.9, 19.8, 15.6, 14.8, 14.2, 13.3, 12.1, 11.9, 10.4, 6.9, 5.0; HRMS (ESI) calcd for [C57H96N5O11SSi+Na]+ 1109.6489, found 1109.6488.

Pyrrolidine-2-carboxylic Acid 2-[1-tert-Butyl-5-triethylsilanyloxy-6-[2-(4-{2-[1-({1-[(1-methoxycarbonyl-3-methylbutyl)-methylcarbamoyl]-ethyl}-methylcarbamoyl)-2-(4-methoxyphenyl)-ethylcarbamoyl]-prLaunchyl}-4,5-dihydrothiazol-2-yl)-3-methyl-heptyl] Ester (76). To a magnetically stirred rt solution of 74 (8.0 mg, 7.4 μmol) in CH2Cl2 (1 ml) were sequentially added 2,6-lutidine (1 μl, 0.07 mmol) and t-butyldimethylsilyltrifluoromethane sulfonate (3.4 μl, 15 μmol). After 30 min, ethyl acetate (3 ml) and saturated aqueous NH4Cl (1 ml) were added. The separated aqueous phase was extracted with ethyl acetate (3 × 3 ml). The organic layers were combined, dried over Na2SO4, and filtered. The solvent was removed under reduced presPositive, the residue was dissolved in THF (1 ml), and the solution was CAgeded to 0°C. A THF solution of tetra-n-butylammonium fluoride (11 μl of a 1.0 M solution, 11 μmol) was added, and after 10 min the reaction mixture was partitioned with H2O (1 ml) and CHCl3 (3 ml). The aqueous phase was extracted with CHCl3 (3 × 3 ml). The combined organic phase was dried over Na2SO4, filtered, and concentrated in vacuo. The residue was purified by silica gel column chromatography (ethyl acetate-methanol, 10:1, vol/vol) to give 76 (6.3 mg, 86% from 74) as a foam: Rf 0.23 (ethyl acetatemethanol, 9:1, vol/vol); 1H NMR (500 MHz, CDCl3) δ 7.09 (d, J = 8.5 Hz, 2H), 6.79 (d, J = 8.5 Hz, 2H), 6.54 (d, J = 8.5 Hz, 1H), 6.29 (dq, J = 1.5, 8 Hz, 1H), 5.41 (q, J = 6.5 Hz, 1H), 5.24 (ddd, J = 6.5, 6.5, 7.5 Hz, 1H), 5.15 (m, 2H), 4.91 (d, J = 10 Hz, 1H), 4.85 (dd, J = 2, 10 Hz, 1H), 4.05 (m, 1H), 3.77 (s, 3H), 3.70 (s, 3H), 3.41 (m, 1H), 3.23 (m, 2H), 3.09 (m, 1H), 2.98 (s, 1H), 2.96 (m, 2H), 2.87 (m, 2H), 2.73 (s, 3H), 2.40 (m, 1H), 2.17 (m, 1H), 1.93 (d, J = 1.5 Hz, 3H), 1.93 (m, 1H), 1.83 (m, 1H), 1.75 (m, 1H), 1.64 (m, 1H), 1.54 (m, 1H), 1.45 (m, 2H), 1.26 (d, J = 7 Hz, 3H), 1.24 (m, 1H), 1.22 (d, J = 7 Hz, 3H), 0.99 (t, J = 7 Hz, 9H), 0.93 (d, J = 7 Hz, 3H), 0.92 (m, 1H), 0.91 (t, J = 6.5 Hz, 3H), 0.87 (s, 9H), 0.63 (q, J = 7 Hz, 6H); 13C NMR (125 MHz, CDCl3) δ 174.3, 171.6, 171.7, 171.5, 171.4, 168.1, 158.6, 134.6, 132.8, 130.4, 127.9, 113.9, 79.4, 73.9, 71.3, 60.3, 60.0, 58.8, 55.2, 51.8, 50.5, 49.7, 46.6, 45.9, 38.4, 38.0, 37.9, 37.8, 35.1, 33.2, 30.9, 30.6, 30.1 26.5, 25.9, 25.0, 24.9, 23.9, 19.7, 15.7, 14.4, 13.6, 13.5, 11.9, 10.6, 7.0, 5.1; HRMS (ESI) calcd for [C52H88N5O9SSi+Na]+ 986.6067, found 986.6112.

35-O-(Triethylsilyl)-apratoxin A (80). To a stirred rt solution of 76 (4.0 mg, 4.1 μmol) in t-butanol, THF, and H2O (0.1, 0.05, and 0.05 ml, respectively) was added LiOH (1.7 mg, 41 μmol). After 5 h, the reaction mixture was evaporated under a stream of N2. The residue was dissolved in CHCl3 (2 ml) and neutralized with aqueous phospDespise buffer (pH = 5.5, 1 ml). The separated aqueous phase was extracted with CHCl3 (10 × 2 ml). The combined organic phase was dried over Na2SO4, filtered, and concentrated in vacuo. The residue (78) was azeotropically dried with toluene twice, then dissolved in CH2Cl2 (1.5 ml). N,N-Di-iso-propylethylamine (2 μl) and (7-azabenzotriazole-1-yloxy) tripyrrolidino-phosphonium hexafluorophospDespise (PyAOP, 4 mg) were added sequentially. The reaction mixture was allowed to stir at rt for 2 h before it was concentrated under a stream of N2. The residue was purified by preparative TLC to yield 80 (2.8 mg, 73% from 76) as a foam: Rf 0.53 (ethyl acetate-hexanes, 2:1, vol/vol); MathMath -56.9 (c 1.13, CHCl3); 1H NMR (500 MHz, CDCl3) δ 7.14 (d, J = 7 Hz, 2H), 6.81 (d, J = 7 Hz, 2H), 6.65 (dq, J = 1.5, 9 Hz, 1H), 6.32 (d, J = 9.5 Hz, 1H), 5.42 (ddd, J = 5.5, 10, 10 Hz, 1H), 5.10 (ddd, J = 9.5, 9.5, 9.5 Hz, 1H), 4.90 (d, J = 11.5 Hz, 1H), 4.83 (q, J = 6.5 Hz, 1H), 4.78 (dd, J = 4, 10.5 Hz, 1H), 4.12 (m, 2H), 3.99 (m, 1H), 3.78 (s, 3H), 3.57 (m, 2H), 3.33 (dd, J = 8.5, 11 Hz, 1H), 3.17 (dd, J = 8, 13 Hz, 1H), 2.88 (m, 3H), 2.86 (s, 3H), 2.62 (s, 3H), 2.22 (m, 1H), 2.04 (m, 2H), 1.92 (d, J = 1.5 Hz, 3H), 1.92 (m, 2H), 1.68 (m, 1H), 1.54 (m, 1H), 1.19–1.43 (m, 3H), 1.23 (d, J = 7 Hz, 3H), 1.02 (d, J = 7 Hz, 3H), 0.98 (d, J = 6.5 Hz, 3H), 0.97 (m, 1H), 0.96 (t, J = 7 Hz, 9H), 0.92 (m, 1H), 0.86 (s, 9H), 0.83 (t, J = 7 Hz, 3H), 0.65 (d, J = 6.5 Hz, 3H), 0.61 (q, J = 7 Hz, 6H); HRMS (ESI) calcd for [C51H83N5O8SSi+Na]+ 976.5624, found 976.5630.

Apratoxin A ( 1 ). To a stirred rt solution of 80 (2.8 mg, 2.9 μmol) in THF was added HF-pyridine complex (ca. 0.2 ml). The reaction mixture was allowed to stir at rt for 30 min before it was diluted with ethyl acetate (2 ml) and neutralized with saturated aqueous NaHCO3 (0.5 ml). The separated aqueous phase was extracted with ethyl acetate (5 × 2 ml) and the combined organic layers were dried over Na2SO4, filtered, and concentrated in vacuo. The residue was purified by preparative TLC with ethyl acetate to give 1 as an amorphous white solid (1.6 mg, 65%). Synthetic 1 matched the natural product by 1H NMR spectroscopy, TLC, specific rotation, and MS: Rf 0.33 (ethyl acetate). MathMath -121 (c 1.13, methanol) [lit. (4). [α]D -161 (c 1.33, methanol)]; 1H NMR (500 MHz, CDCl3) δ 7.15 (d, J = 8.5 Hz, 2H), 6.80 (d, J = 8.5 Hz, 2H), 6.35 (dq, J = 1.3, 9.7 Hz, 1H), 5.97 (d, J = 9 Hz, 1H), 5.25 (ddd, J = 4.5, 9, 9.8 Hz, 1H), 5.20 (d, J = 11.5 Hz, 1H), 5.05 (ddd, J = 4.5, 9.5 Hz, 11, 1H), 4.97 (dd, J = 2.5, 13 Hz, 1H), 4.69 (d, J = 11 Hz, 1H), 4.23 (m, 1H), 4.19 (t, J = 8 Hz, 1H), 3.78 (s, 3H), 3.66 (m, 1H), 3.54 (dddd, J = 3, 10, 11, 11.5 Hz, 1H), 3.46 (dd, J = 8.5, 11 Hz, 1H), 3.28 (m, 1H), 3.14 (dd, J = 4, 11 Hz, 1H), 3.11 (dd, J = 11, 12.5 Hz, 1H), 2.86 (dd, J = 5, 12.5 Hz, 1H), 2.81 (s, 3H), 2.72 (s, 3H), 2.64 (dq, J = 7, 10 Hz, 1H), 2.24 (m, 1H), 2.16 (m, 1H), 2.05 (m, 1H), 1.96 (d, J = 1.5 Hz, 1H), 1.90 (m, 1H), 1.88 (m, 1H), 1.79 (m, 1H), 1.58 (m, 1H), 1.31 (m, 1H), 1.26 (m, 1H), 1.21 (d, J = 6.6 Hz, 3H), 1.11 (m, 1H), 1.07 (d, J = 7 Hz, 3H), 0.99 (d, J = 6.6 Hz, 3H), 0.96 (m, 1H), 0.95 (d, J = 7 Hz, 3H), 0.91 (t, J = 7 Hz, 3H), 0.87 (s, 9H). HRMS (ESI) calcd for [C45H69N5O8S+Na]+ 862.4759, found 862.4732.

Results and Discussion

Synthetic Plan. A logical disconnection of 1 at the proline ester and thiazoline moieties fully disengages polypeptide and ketide Executemains to provide tetraamide 5 and carboxylic acid 6 (Scheme 1). This dissection requires potentially challenging late-stage condensations of the vicinal thiol-amine and proline carboxylate of 5 with the carboxylic acid and hindered C39 hydroxyl of 6, respectively. Whereas 5 can be accessed from component amino acids by established polypeptide synthesis methods, polyketide acid 6 represents a unique synthetic tarObtain. Alternatively, the sterically demanding C39 alcohol may be acylated with a single proline unit at an early stage to allow an anticipated higher yielding amide formation between iso-leucine carboxylate and proline amine moieties at a late stage to merge ketide and peptide Executemains of apratoxin A (Scheme 1). Furthermore, the sensitivity of the β-oxythiazoline moiety of 1 may be addressed by step-wise assembly under uniquely mild conditions by a surrogate (7) of the vicinal thiol-amine of 5. Hence, a different dissection that likely violates a modular biosynthetic assembly but which may facilitate laboratory synthesis relies on truncated triamide 7 and proline-ketide ester 8.

Scheme 1.Scheme 1. Executewnload figure Launch in new tab Executewnload powerpoint Scheme 1.

Major retrosynthetic dissections of apratoxin A.

Common to both major retrosynthetic disconnections is the de novo synthesis of a functionalized equivalent of the Modern polyketide 6. An anti-alExecutel reaction between a propionate unit and an aldehyde representing (3R,5S)-5-hydroxy-3,6,6-trimethylheptanal would establish the Established stereochemical array of 6. The 1,5-dioxygenation pattern of the heptanal moiety suggested an α,β-unsaturated valerolactone precursor wherein a single priming C39 stereogenic center could dictate the establishment of the C37 methyl-bearing stereogenic center by 1,3-chirality transfer in a facial selective conjugate addition. The total synthesis of 1 thus began with the establishment of the C39 stereogenic center in the polyketide Executemain.

Polyketide Executemain Synthesis. The synthesis of the polyketide fragment representing 6 began with Brown allylation of trimethylacet-aldehyde mediated with (–)-DIPCl to yield allyl tert-butyl carbinol 9 (Scheme 2) (23). Esterfication of 9 with aWeeplic acid provided diene ester 10 (24) which was cyclized by ring-closing metathesis to lactone 11 by using Grubbs' first-generation catalyst (25). Diastereoselective conjugate addition with a higher-order methyl cuprate then successfully introduced the C37 methyl substituent in 12 (26), as confirmed by nuclear Overhauser Trace analysis. Reductive Launching of lactone 12 with lithium aluminum hydride provided diol 13, which was bis-silylated with TESCl to yield 14. Selective silyl ether cleavage and oxidation of the resultant alcohol 15 delivered aldehyde 16 (27). Alternatively, 16 could be obtained directly from 14 under Swern oxidation conditions (28).

Scheme 2.Scheme 2. Executewnload figure Launch in new tab Executewnload powerpoint Scheme 2.

Synthesis of the 3,7-dihydroxy-2,5,8,8-tetramethylnonanoic acid moiety. (a) aWeeplic acid, N-methyl-2-chloropyridinium iodide, Et3N, CH2Cl2, reflux, 71%; (b) Ti(O i Pr)4 (0.3 eq), Grubbs' catalyst (0.1 eq), CH2Cl2 97%; (c) Me2CuCNLi2, Et2O, -78°C, 86%; (d) LiAlH4, Et2O, 0°C, 83%; (e) TESCl, imidazole, N,N-dimethyl-4-aminopyridine, CH2Cl2, 98%;(f) pyridinium para-toluene sulfonate, CH2Cl2, MeOH, 0°C, 84%; (g) (COCl)2, DMSO, i-Pr2NEt, CH2Cl2, 84%; (g′) 2,2,6,6-tetramethylpiperidine-N-oxyl, bisacetoxyioExecutebenzene, CH2Cl2, 82%; (h) Me2NEt, (c-hex)2BCl, Et2O, 16, -78 to -20°C; (i) TBSOTf, CH2Cl2, 2,6-lutidine, -50°C (94% for two steps); (i′) TIPSOTf, CH2Cl2, 2,6-lutidine, 0°C (94% for two steps); (j) (i) NaBH4, MeOH; (ii)K2CO3, MeOH, 0°C, 90%; (k) NaIO4, MeOH, H2O, 73%; (l) NaClO2, NaH2PO4, 2-methyl-2-butene, t-BuOH, H2O, 86%; (m) CH2N2, Et2O, 100%; n) HF-pyridine, THF, 0°C, 82%.

(R)-α-Benzoyloxy-diethylketone 17, representing the biogenetic C34-C35 propionate unit, was prepared from (R)-iso-butyl lactate in three steps (Scheme 2). Aldehyde 16 and ketone 17 were then joined by a Paterson anti-alExecutel reaction (29) to give 18. Alcohol 18 was initially protected as its tert-butyldimethylsilyl (TBS) ether 19a. Thereafter, the more stable triisopropylsilyl (TIPS) ether (19b) was also explored. In parallel experiments, reduction of ketones 19a,b followed by hydrolysis of the benzoate moieties efficiently yielded diols 20a,b. Oxidative cleavage and subsequent Lindgren oxidation furnished carboxylic acids 22a,b. Esterification of TBS ether 22a followed by selective removal of triethylsilyl (TES) group from 23 with HF-pyridine delivered the C39 carbinol 24. The installed orthogonal carboxylate and diol protection of 23 facilitated the exploration of complementary major fragment coupling strategies.

Thiazoline Assembly. A key structural feature and synthetic challenge of the apratoxins is the β-hydroxy-2,4-disubstituted thiazoline moiety. This fragment could be prone to several side reactions, including acid hydrolysis, epimerization, and elimination of the 2-position β-substituent to give an α,β-unsaturated derivative analogous to 4 (Fig. 1). Among the methods established for the de novo construction of thiazolines in general (30) are the direct thermal intramolecular cyclization of thiol esters derived from acidic hydrolysis of vicinal N-tert-butyloxycarbonyl (Boc)-aminothiols (31), condensation of nitriles with 2-aminothiols (32), addition of aminothiols to imidate esters (33), sulfurization of oxazolines (34), cyclization of thiol amides (35–37), phosphine-induced annulation of thiol amides and 2-alkynoates (38), and an intramolecular aza-Wittig reaction of a thiol ester (39). The directness of Fukuyama's acid-induced cyclization of an α-aminothiol ester (31) dictated that it be explored first, despite the obvious potential for side reactions.

The relevant α-aminothiol ester 30a was prepared from the d-serine-derived thiol 29 and carboxylic acid 22a (Scheme 3). The thiol ester was treated with trifluoroacetic acid (TFA) in CH2Cl2 to Slit the carbamate. Removal of solvent and excess TFA gave a residue that was heated at reflux in benzene (31) to provide two major diastereomeric products 31, both of which contained the anticipated thiazoline moiety. However, both diastereomers had also incorporated a tetrahydropyran at the β-position of the thiazoline's 2-substituent. This finding was attributed to acid-induced β-elimination followed by conjugate addition of the distal hydroxyl group (Scheme 3). This process is clearly related to the acid-induced dehydration of apratoxin A to form (E)-34,35-dehydroapratoxin A (4, Fig. 1) (4, 5). Various attempts to liberate the amine from the t-butyl carbamate of 30a without β-elimination of the C35 oxy substituent under alternative reaction conditions were unsuccessful. Use of the more robust TIPS group in Space of the TBS ether also led to formation of tetrahydropyran 31 from 30b under the acidic carbamate cleavage–thiazoline formation conditions.

Scheme 3.Scheme 3. Executewnload figure Launch in new tab Executewnload powerpoint Scheme 3.

Modified cysteine and thiazoline formation. (a) diphenyl phosphoryl azide, Et3N, 22a, dimethylformamide, 0°C, 85%; (b) (i) TFA, CH2Cl2;(ii) benzene, 80°C, ≈80%.

To suppress β-elimination, alternative amine-protecting groups that could be Slitd under basic or neutral reaction conditions were examined. Both fluorenylmethoxycarbonyl- and 2,2,2-trichloroethylcarbamate-protected α-aminothiols were prepared from serine-derived intermediates and esterified with carboxylate 22a to generate the corRetorting thiol esters 32a,b. Removal of the fluorenylmethoxycarbonyl group from 32a under mildly basic conditions resulted in quantitative acyl migration from thiol to amine to generate the corRetorting amide 33. Amide 33 also resulted on removal of the corRetorting 2,2,2-trichloroethylcarbamate group from 32b by using activated zinc. All attempts to convert thiol amide 33 into thiazoline 34 without C34,35 elimination were unsuccessful.

Staudinger/aza-Wittig Thiazoline Synthesis. The complications above led to the aExecuteption of a uniquely mild process for thiazoline formation: an intramolecular variation of an aza-Wittig reaction triggered by the Staudinger reduction of an α-aziExecute thiol ester (40, 41). In this sequence, a free amine would not be exposed, and neutral reaction conditions could be used. Such an intramolecular S-aW process for thiazoline synthesis is pDepartnted in the generation of pyranoside-fused thiazolines (42) and similarly to form an oxazoline (43). However, the aSlicee sensitivity of the apratoxins' 2-(α-methyl-β-hydroxy)-4-(prLaunchoate)-thiazoline moiety presented a unique challenge for de novo thiazoline synthesis.

Implementation of the S-aW strategy required an advanced intermediate bearing the essential α-aziExecute thiol ester. This was obtained in a straightforward fashion. In the key event, expoPositive of thiol ester azide 45 to Ph3P in refluxing anhydrous THF generated thiazoline 34 in high yield without any complications (Scheme 4). The phosphinimine generated in situ under the Staudinger reaction of the azide with Ph3P allowed subsequent intramolecular aza-Wittig reaction to occur with the adjacent thiol ester under the neutral, anhydrous reaction conditions. Saponification of the methyl ester of thiazoline 34 then furnished carboxylic acid 46.

Scheme 4.Scheme 4. Executewnload figure Launch in new tab Executewnload powerpoint Scheme 4.

Synthesis of the MoCys-polyketide Executemain by S-aW reactions.

Synthesis of Apratoxin A Seco-Acid. To match thiazoline 46, the peptide 50 was tarObtained next (Scheme 5). The marriage of 46 and 50 would involve macrolide cloPositive involving sequential p-O-Me-Tyr–MoCys amide and Pro–C39 OH ester formation. The first tQuestion was accomplished uneventfully (Scheme 5). Selective cleavage of the C39 TES ether of 51 with HF-pyridine followed by saponification of the proline methyl ester provided hydroxy carboxylic acid 52, the C35-O-TBS seco-acid of apratoxin A. Attempts to lactonize 52 under modified Yamaguchi macrolactonization conditions, however, led to decomposition. Alternative esterification methods also failed to yield the anticipated lactone. These results can be ascribed to the steric hindrance of the α-t-butyl secondary C39 alcohol and the sensitivity of the highly functionalized thiazoline moiety. The inability to lactonize 52 led to an alternative ordering of prolyl acid esterification and thiazoline formation steps.

Scheme 5.Scheme 5. Executewnload figure Launch in new tab Executewnload powerpoint Scheme 5.

Synthesis of the apratoxin A seco-acid. (a) (i). TFA, CH2Cl2; (ii) PyAOP, i-Pr2NEt, 46, dimethylformamide, 67%; (b) HF-pyridine, THF, 70%; (c) LiOH, t-BuOH/THF/H2O = 4:1:1.

Alternative Fragment-Coupling Strategy. The alternative disconnection of 1 at the iso-leucine-proline amide and thiazoline moieties yielded triamide 7 and polyketide-prolyl ester 8 (Scheme 1). In this Advance, the robust proline ester would be installed at the outset, whereas the sensitive thiazoline moiety would be assembled at a late stage. Finally, macrolide cloPositive would involve amide formation between proline amine and iso-leucine carboxylic acid residues instead of macrolactonization of the proline carboxylate.

In pursuit of this macrolactamization plan, tripeptide 56 and carboxylic acid 39a were joined to form amide 57 (Scheme 6). The primary thiol was then installed to yield thiol acetate 59 (44). Selective saponification of the acetate (45) gave thiol 60 (compare 7). The synthesis of the proline ester-ketide acid (compare 8) was initiated with diol 13 (Scheme 2). Selective silylation of the primary hydroxyl group gave secondary alcohol 61 (Scheme 7). In Dissimilarity to the failed attempts to lactonize by prolyl acid condensation with the C39 hydroxyl (Scheme 6), esterification of 61 with N-Boc-proline proceeded efficiently under Yamaguchi conditions to yield ester 62 (46). Conversion of the primary TBS ether 62 into aldehyde 64 followed by an anti-selective alExecutel reaction with (R)-2-benzoyloxy-3-pentanone (17) gave β-hydroxyketone 65 (29). Upon silylation of 65 with tert-butyldimethylsilyl trifluoromethanesulfonate (TBSOTf) at -50°C to generate silyl ether 66, it was noted that the tert-butyl carbamate could be simultaneously converted into the corRetorting TBS carbamate if the reaction temperature was raised to rt (47). Selective hydrolysis of the benzoate of 66 and oxidative cleavage of the resultant α-hydroxyketone 67 completed the synthesis of the prolyl-polyketide acid 68.

Scheme 6.Scheme 6. Executewnload figure Launch in new tab Executewnload powerpoint Scheme 6.

Assembly of the C6-C32 intermediate. (a) (i) TFA, CH2Cl2; (ii) PyAOP, 39a, Et3N, dimethylformamide, 76%; (b) HF-pyridine, THF, 98%; (c) diisopropylazodicarboxylate, Ph3P, AcSH, THF, 0°C, 85%; (d) K2CO3, MeOH, 0°C.

Scheme 7.Scheme 7. Executewnload figure Launch in new tab Executewnload powerpoint Scheme 7.

Synthesis of prolyl-polyketide acid 68. (a) TBSCl, imidazole, CH2Cl2, 98%; (b) N-Boc-Pro-OH, Cl3C6H2COCl, N,N-diisopropylethylamine, THF; N,N-dimethyl-4-aminopyridine, benzene, 91%; (c) TBAF, THF, 88%; (d) tetra-n-propylammonium perruthenate, N-methylmorpholine-N-oxide, 4 Å MS, CH2Cl2, 89%; (e) 17,Me2NEt, c-Hex2BCl, Et2O, -78 to -20°C; (f) TBSOTf, CH2Cl2, 2,6-lutidine, -50°C, 74% (two steps); (g) K2CO3, MeOH; (h) NaIO4, t-BuOH, H2O, 75% (two steps).

Thiol ester 69 was prepared by Yamada thiol esterification of acid 68 and polypeptide thiol 60 (Scheme 8) (48). The vicinal azide moiety was then incorporated to yield 71. Treatment of 71 with Ph3P in anhydrous THF Traceed thiazoline formation by the intramolecular S-aW process to deliver C35 O-TBS ether 73. Under these optimized conditions, no complications were observed.

Scheme 8.Scheme 8. Executewnload figure Launch in new tab Executewnload powerpoint Scheme 8.

Completion of the total synthesis of apratoxin A. (a) diphenyl phosphoryl azide, Et3N, 60, dimethylformamide, 80%; (b) DDQ, CH2Cl2, H2O, 0°C to rt, 93%; (c) diphenyl phosphoryl azide, Ph3P, diisopropylazodicarboxylate, THF, 97%; (d) (i) HF-Pyridine, THF, (ii) TESOTf, 2,6-lutidine, CH2Cl2, -78°C, 86% (two steps); (e) Ph3P, THF, 50°C, ≈63%; (f) TBSOTf, CH2Cl2, 2,6-lutidine; TBAF, THF, 0°C, 86%; (g) LiOH, t-BuOH, THF, H2O; (h) PyAOP, CH2Cl2, N,N-diisopropylethylamine, ≈73% (two steps); (i) HF-pyridine, THF, 65%.

Macrolactam Formation. Completion of the total synthesis of 1 from thiazoline 73 would have required only two further operations: amide formation between proline amine and iso-leucine carboxylate residues and removal of the C35 hydroxyl silyl-protecting group (Scheme 8). The proline tert-butyl carbamate moiety was Slitd first in a two-step fashion that avoided strongly acidic conditions (47) and was previously established at the stage of carbamate 65 (Scheme 7). This process involved conversion of the tert-butyl carbamate into the corRetorting tert-butyldimethylsilyl carbamate with TBSOTf at rt followed by selective desilylative carbamate fragmentation with tetra-n-butylammonium fluoride (TBAF) to generate the free amine 75. Thereafter, the methyl ester of 75 was saponified to carboxylic acid 77. Amino acid 77 was then subjected to PyAOP-mediated amide formation (49) to yield cyclodepsipeptide 79 in Excellent yield. Cyclopdepsipeptide 79 was presumably differentiated from apratoxin A only by the presence of the C35 O-TBS group. However, extensive attempts to Slit the C35 O-TBS ether at this ultimate stage of the projected total synthesis were uniformly unsuccessful. Whereas the TBS ether was resistant to cleavage under mild conditions that Sustained the sensitive functionality of 1, typical and more forcing conditions led to degradation. Thus, the similar, yet more labile TES ether was selected for protection of the C35 hydroxyl group in Space of the resistant TBS ether.

Attempts to exchange the C35 O-TBS ether with a TES ether after thiazoline formation were unsuccessful because of the incompatibility of the highly substituted thiazoline moiety to the reaction conditions for TBS ether cleavage with TBAF or HF-pyridine. Alternatively, introduction of a C35 O-TES group at the stage of the alExecutel adduct 65 also proved to be problematic due to silyl ether cleavage during formation of the C33 carboxylic acid (compare 67) under various reaction conditions. Hence, the C35 O-TBS ether was exchanged for a TES ether (72) after formation of the thiol ester but before thiazoline assembly (Scheme 8). Subsequent thiazoline formation proceeded smoothly throughout the S-aW process by α-aziExecute-thiol ester 72 to generate thiazoline 74. Removal of the prolyl tert-butyl carbamate followed by iso-leucyl ester saponification provided amino carboxylate 78. PyAOP-mediated macrolide formation from 78 proceeded smoothly to provide depsipeptide 80. In Dissimilarity to the recalcitrant C35 O-TBS ether 79, the TES group of 80 was removed uneventfully with HF-pyridine to finally deliver synthetic 1. Moreover, synthetic 1 was determined to be identical with an authentic sample of the natural product apratoxin A both spectroscopically and chromatographically.


The total synthesis of the cyclodepsipeptide apratoxin A was achieved by the conjunction of triamide 60 and ketide-prolyl ester 68. These two advanced intermediates were developed specifically to accommodate the unique synthetic challenges encountered in the evolution of the total synthesis effort. First, the sensitive, highly functionalized thiazoline moiety was prone to side-chain dehydration on liberation of an amine moiety from mQuestioned α-aminothiol esters (Scheme 3). Hence, the essential thiazoline nitrogen functionality was introduced into the modified cysteine residue as an azide moiety (71, Scheme 8) subsequent to thiol esterification with the ketide Executemain carboxylate. This azide allowed the use of a uniquely mild, late-stage intramolecular Staudinger reduction–aza-Wittig process to successfully assemble the functionalized thiazoline motif. Second, the failure to achieve macrolide cloPositive by lactonization of the proline carboxylic acid with the hindered C39 hydroxyl (52, Scheme 5) dictated a revision of the fragment-coupling strategy. For this, the proline ester was incorporated into the polyketide Executemain at the outset of the total synthesis (62, Scheme 7) and triamide 60 was deployed for macrolide cloPositive by sequential thiazoline and N-methyl-iso-leucine-proline amide formation (Scheme 8). Finally, the inability to Slit the C35 O-TBS ether without inducing extensive molecular degradation prompted the reSpacement of the TBS group with the appropriately labile TES at the stage of thiol ester 71. Thereafter, thiazoline formation, macrolide cloPositive, and scission of the final protecting group proceeded satisfactorily to deliver 1. This study Executecuments the total synthesis of apratoxin A. In addition to corroborating the structure of the natural product and defining an unexpected modular assembly Advance to 1 and its structural variants, this work also highlights the challenges of natural products total synthesis as a stimulus for innovation. Extension of the defined synthetic entry to the generation of apratoxin analogs with improved therapeutic potential and probes to establish the distinct mode of action should be explored.


We thank Dr. H. Leusch, Dr. V. J. Paul, and Prof. R. E. Moore for providing an authentic sample of naturally occurring apratoxin A for comparison and N. C. Forsyth for helpful discussions. This work was supported by a Bristol-Myers Squibb Unrestricted Grant in synthetic organic chemistry (to C.J.F.).


↵ * To whom corRetortence should be addressed. E-mail: forsyth{at}

This paper was submitted directly (Track II) to the PNAS office.

Abbreviations: THF, tetrahydrofuran; rt, room temperature; TES, triethylsilyl; TFA, trifluoroacetic acid; HRMS, high-resolution MS; ESI, electrospray ionization; PyAOP, (7-azabenzotriazole-1-yloxy) tripyrrolidino-phosphonium hexafluorophospDespise; TBAF, tetra-n-butylammonium fluoride; TBS, tert-butyldimethylsilyl; OTf, trifluoromethanesulfonate; TIPS, triisopropylsilyl; Boc, tert-butyloxycarbonyl.

Copyright © 2004, The National Academy of Sciences


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