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Synthesis and Biological Activities of Aplyronine A Analogues toward the Development of Antitumor Protein–Protein Interaction Inducers between Actin and Tubulin: Conjugation of the C1–C9 Macrolactone Part and the C24–C34 Side Chain

Kentaro Futaki
Kentaro Futaki
Graduate School of Pure and Applied Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba 305-8571, Japan
More by Kentaro Futaki

Momoko Takahashi
Momoko Takahashi
Graduate School of Pure and Applied Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba 305-8571, Japan
More by Momoko Takahashi

Kenta Tanabe
Kenta Tanabe
Graduate School of Pure and Applied Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba 305-8571, Japan
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Akari Fujieda
Akari Fujieda
Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa, Nagoya 464-8601, Japan
More by Akari Fujieda

Hideo Kigoshi*
Hideo Kigoshi
Graduate School of Pure and Applied Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba 305-8571, Japan
*E-mail: [email protected] (H.K.).
More by Hideo Kigoshi
http://orcid.org/0000-0003-1554-5129

, and

Masaki Kita*
Masaki Kita
Graduate School of Pure and Applied Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba 305-8571, Japan
Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa, Nagoya 464-8601, Japan
*E-mail: [email protected] (M.K.).
More by Masaki Kita
http://orcid.org/0000-0001-6685-4461

Cite this: ACS Omega 2019, 4, 5, 8598–8613

Publication Date (Web):May 16, 2019

https://doi.org/10.1021/acsomega.9b01099

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Abstract

Aplyronine A (ApA) is an antitumor marine macrolide that induces an protein–protein interaction (PPI) between actin and tubulin. The C1–C9 macrolactone part including the C7 N,N,O-trimethylserine (TMSer) ester is important for its highly potent activities. To develop new antitumor PPI inducers, four aplyronine analogues were synthesized, which bear the C1–C9 macrolactone part with 0–2 TMSer ester(s) and the C24–C34 actin-binding side chain. Despite exhibiting potent actin-depolymerizing activity comparable to that of ApA, these analogues did not show potent cytotoxicity or depolymerize microtubules. Molecular modeling studies suggested that the whole macrolactone moiety of aplyronines was important to fix the conformation of the C7 TMSer ester moiety, while the linear C1–C9 part was insufficient. Still, our study newly proposed that fixed conformations of the C7 or C9 TMSer esters in aplyronines that protrude from the actin surface are important for binding to tubulin and inhibit microtubule dynamics.

Introduction

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The manipulation of new protein–protein interactions (PPIs) by natural products and related small bioactive compounds has become an important issue in the fields of basic life sciences and drug discovery.(1) The use of PPI-stabilizing or -inhibiting scaffolds has important implications for simplified mimetic design and provides new insights into ways to design molecules that tether proteins using different binding surfaces.(2) Among a small number of natural products that simultaneously interact with more than one biomacromolecule, aplyronine A (ApA, 1),(3−5) a 24-membered antitumor macrolide isolated from the sea hare Aplysia kurodai, promotes a unique PPI between actin and tubulin, two of the major cytoskeletal proteins (Figure 1). ApA was originally characterized as an actin-targeting agent that depolymerizes fibrous actin (F-actin) and inhibits the polymerization of actin by forming a 1:1 complex with the monomeric globular molecule (G-actin).(6) While it also exhibits potent cytotoxicity against HeLa S3, a human cervical carcinoma cell line at an IC₅₀ of 10 pM, this concentration is much lower than that needed for disassembly of the actin cytoskeleton (∼100 nM). We recently showed that ApA forms a 1:1:1 heterotrimeric complex with actin and tubulin, in association with the synergistic binding of actin to tubulin, and inhibits tubulin polymerization.(7) This synergistic binding effect of the actin–ApA complex toward tubulin has been supported through the use of gel-permeation HPLC, surface plasmon resonance analysis,(8) and photolabeling and affinity pulldown experiments.(9,10) Several tubulin-targeting agents have been widely used in cancer chemotherapy. To the best of our knowledge, however, ApA is the first microtubule inhibitor that has been shown to have a PPI-inducing effect between actin and tubulin to inhibit microtubule dynamics.(2)

Figure 1. Structures of aplyronines and their synthetic analogues.

The structure of ApA can be divided into two characteristic parts: the C1–C23 macrolactone with an N,N,O-trimethylserine (TMSer) ester and the C24–C34 side chainwith an N,N-dimethylalanine (DMAla) ester, and the latter is important for actin-depolymerizing activity.(11) X-ray analysis of the actin–ApA complex revealed that ApA intercalates into the hydrophobic cleft between subdomains 1 and 3 of actin by using the side-chain moiety of 1.(12) The crystallographic structures of complexes of actin with several macrolides that have side chain(s) similar to those of ApA have also been reported, such as kabiramide C,(13) sphinxolide B,(14) reidispongiolides A and C,(14) and swinholide A,(15) all of which bind to actin at the same site. In fact, a synthetic ApA analogue of the C21–C34 side-chain 4 specifically binds to actin at the same site as aplyronines and moderately depolymerizes F-actin.(16) Meanwhile, as for the macrolactone moiety, the presence of the C7 TMSer ester in 1 is essential for the potent activity of ApA because aplyronine C (ApC, 3), which lacks the C7 TMSer ester, does not bind to tubulin and its cytotoxicity is 1700 times weaker than that of 1.(9b) Thus, it has been suggested that the positively charged C7 TMSer ester that protrudes from the actin–ApA complex might generate a unique tubulin-binding site, which drives the interaction with tubulin α/β-heterodimer.(7)

ApA exhibits potent antitumor activities in vivo against P388 leukemia, Lewis lung carcinoma, and Ehrlich carcinoma.(3a) To develop new PPI modulators with potent biological activities, two ApA analogues have been synthesized, which bear the whole macrolactone moiety of ApA and the side-chain parts of mycalolide B or swinholide A.(17) Both of these “hybrid” molecules exhibited potent actin-depolymerizing activity, and the latter showed potent cytotoxicity and induced potent PPI between actin and tubulin, as with ApA. Meanwhile, there have been few studies on the structural modification and simplification of the macrolactone moiety of aplyronines. To better understand the binding modes and antitumor mechanism of ApA and to develop new analogues that retain the potent PPI-inducing activity of ApA between actin and tubulin as new drug leads, we examined here the simplification of the macrolactone part of aplyronines.

Results and Discussion

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To get perspectives for the design of structurally simplified aplyronine analogues, molecular modeling studies were performed. Aplyronine B (ApB, 2) is a minute congener of ApA isolated from A. kurodai, in which the TMSer ester is migrated from C7 to C9. It is less cytotoxic than ApA but 7- to 8-fold more cytotoxic than ApC.(3a,4c) While the crystallographic structures of complexes of ApB or ApC with actin have not been reported, they are expected to bind to actin, similar to ApA and related actin-depolymerizing macrolides. Thus, conformational searches for ApA and ApB bound to actin were performed using the Amber12:EHT force-field, in which both actin and the C24–C34 side chainwere fixed. Among the ten and two lowest-energy conformers of ApA and ApB obtained within 30 kJ/mol, those with the lowest energies were almost dominant (97.8 and 99.9% occupations, respectively) (Figure 2). In the most stable conformer of ApA, the macrolide part (C1–C23) including the C7 TMSer ester moiety almost completely overlapped than those in the actin–ApA complex [PDB: 1WUA] [root-mean-square deviation (rmsd) 0.477 Å for all of the C, O, and N atoms of the macrolactone part]. It was expected that our modeling studies are applicable to precisely predict the conformations of natural and synthetic ApA analogues on actin. In the most stable conformer of ApB, the orientation of the macrolide part was far different from that in the actin–ApA complex, while its C9 TMSer ester protruded from the surface of actin, like the C7 TMSer ester of ApA. While it is still unclear whether ApB interferes with microtubule dynamics in association with actin like ApA, we assumed that some spatial and conformational variance of the TMSer ester might be allowed for PPI-inducing activity and potent cytotoxicity of aplyronines.

Figure 2. Molecular modeling studies of aplyronines. The most stable conformers of ApA [(a), green] and ApB [(b), cyan] on actin are shown. Conformational searches were performed using the Amber12:EHT force-field, in which both actin and the C24–C34 side-chain parts of aplyronines were fixed. In each model, ApA (yellow) in the actin–ApA complex (PDB code: 1WUA) is superimposed.

Previous structure–activity relationship (SAR) studies showed that the functional groups of ApA that are important for cytotoxicity are the C7 TMSer ester, the C9 hydroxy group, and a conjugated diene moiety on the macrolactone ring. Based on the modeling studies as mentioned above, we designed structurally simplified ApA analogue 5, in which the C1–C9 macrolactone part was connected with the C24–C34 side chainvia an ester bond (Figure 1). We expected that the conjugated diene moiety in 5 would partially fix the conformation of the linear C1–C9 part, which makes its C7 TMSer ester direct to the desired position on the complex with actin. For reference, we planned to synthesize ApC analogue 6, which lacks the C7 TMSer ester of 5. We were concerned that 5 might not show potent PPI-inducing activity if the C7 TMSer ester is tightly attached to the actin surface. For such an undesired interaction to occur, bis-C7,C9-TMSer analogue 7 was designed, in which either at least the C7 or C9 TMSer ester would be apart from the surface of actin and interact with tubulin. In addition, to simplify the synthetic pathway, symmetrical bis-C7,C7′-TMSer analogue 8 was designed. All four aplyronine analogues could be prepared via the esterification of C1–C9 (or C7) unsaturated carboxylic acids with the common C23–C34 primary alcohol, followed by regioselective esterification of the TMSer group.

First, the C23–C34 side-chain part 16 was synthesized (Scheme 1). A known primary alcohol 9(18) prepared from (S)-3-hydroxy-2-methylpropionate was converted into the phenyl tetrazole (PT) sulfide with aryl disulfide/ⁿBu₃P, and subsequent oxidation with meta-chloroperbenzoic acid (m-CPBA) yielded PT–sulfone 10. Julia–Kocienski olefination(19) of 10 with sterically hindered aldehyde 11(4c) and lithium hexamethyldisilazide (LHMDS) as a base in 1,2-dimethoxyethane (DME)(20) at temperatures of −55 °C to room temperature afforded an olefin quantitatively (E/Z = 1:2). Simultaneous catalytic hydrogenation of the olefin and hydrogenolysis of the benzyl group using palladium(II) hydroxide on carbon resulted in the epimerization of C29 secondary alcohol. Thus, removal of the benzyl group with calcium-mediated Birch reduction and selective protection of the primary alcohol with a tert-butyldiphenylsilyl (TBDPS) group gave silyl ether 12. Catalytic hydrogenation of the olefin with Pd(OH)₂ on carbon, condensation with N,N-dimethyl-l-alanine, acidic hydrolysis of the C34 methyl acetal, and reduction of the hemiacetal using sodium borohydride afforded diol 13. Trityl group (Tr) protection of the primary alcohol and subsequent acetylation of the remaining C31 secondary alcohol gave acetate 14. Removal of the Tr group with formic acid in ether and oxidation of the primary alcohol with Dess–Martin periodinane(21) provided aldehyde 15. Finally, dehydrating condensation with N-methylformamide under acidic conditions and selective deprotection of the TBDPS group with ammonium fluoride furnished compound 16.

Scheme 1. Synthesis of the C23–C34 Side-chain Part 16^a

We next set out to synthesize three kinds of unsaturated carboxylic acids, which correspond to the C1–C9 or C1–C7 macrolactone part of aplyronines (Scheme 2). Homoallylic alcohol 17,(22) prepared from methyl (S)-3-hydroxy-2-methylpropionate using Hosomi–Sakurai allylation(23) as a key step, was converted to the diol by calcium-mediated Birch reduction. Subsequent acetonide protection of the 1,3-diol, Lemieux–Johnson oxidation of the terminal olefin,(24) and Horner–Wadsworth–Emmons reaction of the aldehyde with triethyl 4-phosphonocrotonate gave conjugated ester 18 with high stereoselectivity (4E/4Z = 10:1). Next, removal of the acetonide group under acidic conditions and selective protection of the primary alcohol with a TBDPS group gave mono-silyl ether 19. To prepare the ApA and ApC analogues 5 and 6 having or lacking the C7 TMSer ester, the C7 hydroxy group was protected with a methylthiomethyl (MTM) ether using dimethylsulfoxide (DMSO)/Ac₂O/AcOH(25) or a tert-butyldimethylsilyl (TBS) ether. Hydrolysis of the ethyl esters under mild conditions using lithium hydroxide provided MTM- and TBS-protected unsaturated carboxylic acids 20 and 21. Similarly, another unsaturated carboxylic acid 23 having the bis-C7,C7′-TBS ether was synthesized from a known aldehyde 22(26) in 2 steps.

Scheme 2. Synthesis of Conjugated Carboxylic Acids 20, 21, and 23^a

With the side-chain and the linear C1–C9 (or C1–C7) macrolactone parts in hand, we examined the synthesis of aplyronine analogues 5–8 (Scheme 3). Condensation of the side-chain part 16 with carboxylic acid 20 using the procedure of Yamaguchi et al.(27) and removal of the MTM group by silver(I) nitrate yielded C7-hydroxy ester 24. Condensation of the secondary alcohol with N,N,O-trimethyl-l-serine and removal of the two silyl groups at C9 and C25 by the hydrogen fluoride–pyridine (HF·py) complex furnished ApA analogue 5. Also, direct removal of the two silyl groups in 24 yielded the corresponding ApC analogue 6 (32% in 5 steps from 19). Because of the low efficiency of the protection/deprotection sequence using an MTM group, we also examined the condensation of 16 with the TBS-protected carboxylic acid 21. Subsequent removal of three silyl groups by HF·py complex at once afforded 6 in a more efficient manner (57% in 4 steps from 19). To our delight, chemoselective condensation at the C7 and C9 hydroxy groups in 6 with N,N,O-trimethyl-l-serine selectively provided bis-C7,C9-TMSer analogue 7, due to the structural hindrance at the C25 secondary alcohol. Finally, condensation of 16 with carboxylic acid 23 followed by the removal of three TBS groups gave triol 25, which was converted to bis-C7,C7′-TMSer analogue 8 as in the case with 7.

Scheme 3. Synthesis of Aplyronine Analogues **5–8**^a

The biological activities of aplyronine analogues 5–8 were compared to those of natural ApA. For the in vitro F-actin sedimentation assay, an ultracentrifugation method was used, which is applicable for various actin-targeting agents and their fluorescent, photoreactive, and other derivatives.(9a,28) The amount of F-actin (3 μM as monomer) in the precipitate (P) fraction was reduced by treatment with ApA in a dose-dependent manner, and the protein bands were observed almost entirely in the supernatant (S) fraction at 5 μM, as with the G-actin (Figure 3a, far left, without Mg²⁺). The EC₅₀ of ApA was 1.4 μM against 3 μM actin, which well coincided with those by a pyrene-labeled (pyrenyl) actin method (EC₅₀ 1.3–1.6 μM against 3–3.7 μM actin)(17) (Table 1). Similarly, four aplyronine analogues 5–8 showed significant actin-depolymerizing activities, with EC₅₀ values of <5, <5, 2.1, and 1.3 μM against 3 μM actin, respectively, all of which were stronger than that of the C21–C34 side-chain analogue 4 (EC₅₀ 7.9 μM against 3.7 μM actin, 20% residual activity of ApA).(16a)

Figure 3. In vitro F-actin and microtubule sedimentation assay. (a) Filamentous (F-) actin (3 μM as a monomer) was precipitated by ultracentrifugation after treatment with aplyronine analogues. (b) Tubulin (3 μM as a heterodimer) was polymerized with paclitaxel (6 μM) in the presence of actin (3 μM) and/or aplyronine analogues and then precipitated by ultracentrifugation. Proteins in the supernatant (S) and the precipitate (P) were analyzed by SDS-PAGE and detected with CBB stain.

Table 1. Biological Activities of ApA and Its Derivatives

		actin-depolymerizing activitya EC₅₀ (μm)
compound	cytotoxicity (HeLa S3) IC₅₀ (nm)	ultra-centrifugation method	pyrenyl actin method
ApA (1)	0.010c [0.45]d^,e	1.4	1.3c [1.6]f^,g
ApB (2)	[2.9]d^,e	b	b
ApC (3)	17c [22]d^,e	b	1.4c
4	[−19 000]e^,f	b	[7.9]f^,g
5	1400	<5	b
6	1100	<5	b
7	1800	2.1	b
8	1900	1.3	b

^{^a}

Values indicate the concentrations required to depolymerize F-actin (3 μM for monomer) to 50% of its control amplitude. Averages of two reproducible runs are shown.

^{^b}

Not examined.

^{^c}

See ref (9b).

^{^d}

See ref (4c).

^{^e}

Original IC₅₀ values are described in ng/mL, and the data calculated as free salts are shown in square brackets.

^{^f}

See ref (16a).

^{^g}

The data using 3.7 μM for monomer actin are shown in square brackets.

The PPI-inducing effects of aplyronine analogues between actin and tubulin were then examined by using a similar ultracentrifugation method. ApA (8.5 μM) specifically induced microtubule disassembly in the presence of actin (3 μM for monomers), and both of these proteins were dominantly detected in the S fraction (Figure 3b).(8) Meanwhile, even at the highest available concentrations of ApA analogue 5 (27 μM) and bis-TMSer analogues 7 and 8 (85 μM each), most of the tubulin remained in the P fractions (ca. 96.8, 87.8, and 92.1%), while substantial actin was moved to the S fractions (ca. 66.9, 84.9, and 87.0%). These results suggested that compounds 5, 7, and 8 scarcely depolymerized microtubules synergistically with actin, similar to ApC.(7)

As for cytotoxicity, four aplyronine analogues 5–8 all moderately inhibited the proliferation of HeLa S3, a human cervical carcinoma cell line (IC₅₀ 1.1–1.9 μM) (Table 1). ApA analogue 5 was >13 times more cytotoxic than side-chain analogue 4, but 140 000- and 82-fold less cytotoxic than natural ApA and ApC, respectively. Furthermore, the cytotoxicities of compounds 5 and 6 were almost the same, which did not reflect the significant difference in the cytotoxicity of natural aplyronines. Previous studies showed that fluorescent aplyronine derivatives, for example, those conjugated with tetramethylrhodamine, are rapidly accumulated in the cytoplasm and cause rapid disassembly of the actin cytoskeleton in HeLa S3 cells, and thus natural aplyronines are highly cell-permeable.(28) However, it is likely that the cell-permeability of the four aplyronine analogues 5–8 is very low, as is that of the side-chain analogue 4, and the presence of positively charged TMSer and/or DMAla esters contributed little to their potent cytotoxicities. As a result, an ApA analogue 5 and bis-TMSer analogues 7 and 8 were unable to induce PPIs between actin and tubulin either in vitro or in cells. Still, however, the C1–C9 part in 5–8 might contribute to the potentiation of actin-depolymerizing activity and cytotoxicity of the C24–C34 side chainby enhancing the interaction with actin.

To consider why synthetic aplyronine analogues 5, 7, and 8 did not show significant PPI-inducing effects between actin and tubulin, molecular modeling studies were performed, in which both actin and the C24–C34 side chainwere fixed. Among the 17 lowest-energy conformers of 5 on actin obtained within 30 kJ/mol, that with the lowest energy accounted for a relatively high proportion (90.3% occupation) (Figure 4a). However, the C1–C9 moiety of the most stable conformer of 5 had a far different conformation from that of ApA (rmsd 4.978 Å for all of the C, O, and N atoms of the C1–C9 part) due to the hydrogen bond between the methoxy group of the C7 TMSer ester and the carboxyl group of Asp25 (Figure 4b), which was not observed in the original actin–ApA complex. In contrast, the C1–C9 moiety of the fourth stable conformer of 5 (ΔE +11.9 kJ/mol, 0.7% occupation) well coincided with that of ApA (rmsd 0.865 Å for all of the C, O, and N atoms of the C1–C9 part) (Figure 4c), in which the C7 TMSer ester protruded from the actin surface, and the C9 hydroxy group directly interacted with the carboxyl group of Glu334 (Figure 4d). Due to such a low proportion of the desired conformation on actin, however, ApA analogue 5 might be unable to interact with tubulin.

Figure 4. Molecular modeling studies of the synthetic analogues of aplyronines. Conformational searches were performed as mentioned in Figure 2. (a–d) The most stable (green) and 4th stable (cyan, ΔE +11.9 kJ/mol) conformers of ApA analogue 5. Selected atom numbers are shown in blue. Two amino acid residues (Asp25 or Glu334) that interact with 5 in (a,c) are shown as sphere models in (b,d), respectively. (e,f) The most stable conformers of bis-TMSer ester analogues 7 (orange) and 8 (magenta) on actin. (g) The actin–ApA complex viewed from the bottom of the macrolide moiety. The Arg147 residue that interacts with the C13–OMe group is shown as a sphere model.

As for the bis-TMSer analogues 7 and 8, 46 and 54 lowest-energy conformers were obtained within 30 kJ/mol. The most stable conformers of 7 and 8 accounted for 89.1 and 71.9%, respectively, and both of their C7 TMSer esters interacted with Asp25, as with 5. While the additional C9 TMSer ester in 7 did not attach to the actin surface, its orientation was far different from those of ApA and ApB models (Figure 4e). In addition, the second C7′ TMSer ester in 8 was still tightly bound to actin (Figure 4f). These undesired major conformations of bis-TMSer esters might explain why 7 and 8 did not exhibit significant PPI-inducing activities.

We considered again the conformation of ApA on actin, and the C13 methoxy group interacts with Arg147 via water molecules in addition to direct interaction of the C9 hydroxy group with Glu334 (Figure 4g). While the conformation of the macrolactone moiety of ApB on actin was far different from that of ApA, its C13 methoxy group was close to Arg147 based on the similar hydrogen bonds with that of the actin–ApA complex (Figure 2b). These results suggested that such multiple hydrogen bonds might be essential to restrict the conformation of the macrolactone moiety of aplyronines and that the linear C1–C9 macrolactone part was insufficient to enable the C7 or C9 TMSer esters to protrude from the actin surface to bind to tubulin, like natural ApA and ApB, and interfere with microtubule dynamics in association with actin.

Conclusions

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For the development of new molecules to regulate PPI between actin and tubulin, four aplyronine analogues, which bear the C1–C9 macrolactone part with 0–2 TMSer ester(s) and the C24–C34 actin-binding side chain, were synthesized with the aid of molecular modeling studies. Despite their actin-depolymerizing activity comparable to that of ApA, these aplyronine analogues did not show significant PPI-inducing effects. Molecular modeling studies suggested that the whole C1–C23 macrolactone moiety of aplyronines was important to fix the conformation of the C7 TMSer ester to interact with tubulin. As a result, our synthesized analogues were oversimplified, and the linear C1–C9 macrolactone and the C24–C34 side-chain parts of aplyronines were insufficient for both tubulin recruitment and potent cytotoxicity. Still, our SAR and molecular modeling studies newly proposed that the C7 or C9 TMSer esters in aplyronines A and B that protruded from the actin surface are important for their potent activities, which are superior to those of other agents that merely target actin, from the viewpoint of antitumor drug discovery and development. Actin is the most abundant protein in eukaryotic cells and is essential for the regulation of various cellular functions, such as cell division and tumor migration. It is possible that actin-targeting agents interact with various cellular targets via unknown PPIs.(2) Thus, the results of this study have potential in the design and development of newly classified PPI modulators as pharmacological tools and therapeutic agents. Further investigations on the mechanisms of action of aplyronines and the development of new PPI-regulating molecules inspired by diverse actin-targeting natural products are currently underway.

Experimental Sections

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General Information

All chemicals were obtained commercially unless otherwise noted. Organic solvents and reagents for moisture-sensitive reactions were distilled by the standard procedure. Anhydrous CH₂Cl₂, tetrahydrofuran (THF), DME, benzene, pyridine, DMSO, and N,N-dimethylformamide (DMF) were obtained commercially. Column chromatography was performed using silica gel BW-820MH or FL60D (75–200 or 45–75 μm, Fuji Silysia Co., Aichi, Japan) or a Yamazen preparative silica gel (40 μm). All moisture-sensitive reactions were performed under an atmosphere of argon or nitrogen, and the starting materials were azeotropically dried with benzene before use. Merck precoated silica gel 60 F254 plates were used for thin layer chromatography.

Spectroscopic Analysis

¹H and ¹³C nuclear magnetic resonance (NMR) spectra were recorded on a Bruker BioSpin AVANCE 600 spectrometer (600 MHz for ¹H and 150 MHz for ¹³C) or AVANCE 400 spectrometer (400 MHz for ¹H and 100 MHz for ¹³C). Chemical shifts are reported in parts per million (ppm) with coupling constants (J) in hertz relative to the solvent peaks, δ_H 7.26 (residual CHCl₃) and δ_C 77.0 for CDCl₃, respectively. Optical rotations were measured with a JASCO DIP-1000 polarimeter using the sodium D line. Infrared (IR) spectra were recorded on a JASCO FT/IR-230 spectrometer. High-resolution electrospray ionization mass spectra (HR-ESIMS) were measured on an AccuTOF CS spectrometer (JEOL).

Cell Culture and Cytotoxicity Assay

HeLa S3 cells (suspension culture-adapted human cervical carcinoma cell line, ATCC CCL-2.2) were cultured in Eagle’s minimal essential medium supplemented with fetal bovine serum (10%) in a humidified atmosphere containing CO₂ (5%), as described previously.(7,10) ApA (1) was isolated from the sea hare A. kurodai, according to published methods.(3,5) For bioassays, ApA and its synthetic derivatives were stored in DMSO at 1–10 mM. The cytotoxicity of ApA and its derivatives was measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) method.(29) In brief, HeLa S3 cells were seeded at 2 × 10³ cells per well in 96-well plates. After incubation overnight at 37 °C, aplyronines (1 pM to 1 mM) were added, and the cells were incubated for 96 h at 37 °C. A 1.4 mg/mL MTT solution in phosphate buffer saline (50 μL) was added to the cells. After 4 h, the culture medium was removed and the formazan product was dissolved in DMSO (150 μL). Optical density at 540 nm was measured with a TECAN microplate reader (Infinite 200 Pro). All assays were performed in duplicate to confirm reproducibility.

In Vitro F-Actin Sedimentation Assay(9a,28)

To a solution of rabbit skeletal muscle actin (3 μM, cytoskeleton) in G-buffer [2 mM Tris·HCl (pH 8.0), 0.2 mM CaCl₂, 0.2 mM ATP, 0.5 mM 2-mercaptoethanol] (500 μL) was added a 0.15 M solution of MgCl₂ (3.3 μL), and the mixture was stirred at 25 °C for 1 h. To the polymerized F-actin solution (200 μL) were added samples (1 μL in DMSO), and the resulting mixtures were stirred at 25 °C for 30 min and then ultracentrifuged (150 000g, 22 °C, 1 h). Aliquots of the supernatants and precipitates (re-dissolved in G-buffer) were mixed with the same amount of 2× SDS buffer (Sigma) and boiled at 95 °C for 5 min. SDS-PAGE was performed by using a precast 10% polyacrylamide gel (ATTO), and the gels were stained with a Quick-CBB kit (Wako). Densitometry of CBB-stained proteins was performed using ImageJ software.

Microtubule Sedimentation Assay(8)

A solution of 6 μM tubulin (as a heterodimer) in modified RB buffer [100 mM PIPES·Na (pH 6.9), 1 mM EGTA, 2 mM MgCl₂] was ultracentrifuged (150 000g, 30 min at 4 °C) to remove oligomeric tubulins. To the supernatants (50 μL) were added 2 mM paclitaxel in DMSO (0.3 μL) to induce microtubule formation, and/or 6 μM actin in modified RB buffer (50 μL) and ApA or its analogues (1–2 μL). After incubation for 30 min at 37 °C, samples were ultracentrifuged (150 000g, 37 °C, 1 h). Aliquots of the supernatants and precipitates (re-dissolved in modified RB buffer) were mixed with the same amount of 2× SDS buffer and boiled for 5 min at 95 °C. SDS-PAGE was performed using a precast 10% polyacrylamide gel, and the gels were stained with a Quick-CBB kit (Wako).

Molecular Modeling Studies

Molecular modeling studies were performed using the Molecular Operating Environment (MOE) 2014.09 program package (Chemical Computing Group, Inc.), as described previously.(10,30) For docking model studies, all water molecules associated with the actin–ApA complex (PDB: 1WUA) were removed, except for those near the ligand, and all protons on the protein and the ligand were complemented. Conformational searches were performed using the Amber12:EHT force-field with GB/VI Generalized Born(31) implicit solvent electrostatics (D_in = 1, D_out = 80) and with LowModeMD,(32) in which both the actin and the side-chain moiety (C23–C34) of aplyronines and their synthetic analogues were fixed.

Synthesis and Spectroscopic Data of ApA Analogues

Thioether 9a

To a stirred solution of primary alcohol 9 (7.1 g, 20 mmol)(18) and 5,5′-dithiobis(1-phenyl-1H-tetrazole) (9.3 g, 26 mmol) in dry THF (67 mL) cooled at 0 °C was added tri-n-butylphosphine (8.0 mL, 32 mmol), and the resulting solution was stirred at room temperature for 16 h. Water (80 mL) was added, and the resulting mixture was extracted with EtOAc (25 mL × 3). The combined extracts were washed with sat. NaHCO₃ aq and brine, dried with Na₂SO₄, and concentrated. The crude material was purified with a SiO₂ column (50 g, hexane/EtOAc = 40/1 to 9/1) to give thioether 9a (9.5 g, 93%) as a light yellow oil. 9a: R_f = 0.78 (1:1 hexane/EtOAc); [α]_D²⁵ −9.7 (c 1.6, CHCl₃); IR (CHCl₃): 3010, 2958, 2930, 2884, 2858, 1598, 1500, 1388, 1253, 1090, 1028, 838, 671 cm^–1; ¹H NMR (600 MHz, CDCl₃): δ 7.59–7.53 (m, 5H), 7.32–7.31 (m, 5H), 4.48 (AB quart, J = 8.3 Hz, 2H), 3.71 (dd, J = 13.0, 4.1 Hz, 1H), 3.66 (dd, J = 5.9, 3.5 Hz, 1H), 3.55 (dd, J = 9.2, 5.4 Hz, 1H), 3.36 (dd, J = 9.2, 6.7 Hz, 1H), 3.17 (dd, J = 13.0, 8.9 Hz, 1H), 2.21 (m, 1H), 2.07 (dddq, J = 6.7, 5.9, 5.4, 6.7 Hz, 1H), 1.07 (d, J = 6.7 Hz, 3H), 1.06 (d, J = 6.7 Hz, 3H), 0.88 (s, 9H), 0.05 (s, 6H); ¹³C NMR (150 MHz, CDCl₃): δ 155.1, 138.8, 134.0, 130.2, 129.9 (2C), 128.5 (2C), 127.8 (2C), 127.6, 124.0 (2C), 77.6, 73.2, 72.7, 38.4, 36.5, 36.0, 26.3 (3C), 18.6, 17.9, 14.9, −4.0 (2C); HRMS (ESI) m/z: 535.2531 (calcd for C₂₇H₄₀N₄NaO₂SSi [M + Na]⁺, Δ −0.8 mmu).

Phenyltetrazole Sulfone 10

To a stirred solution of thioether 9a (1.9 g, 3.7 mmol) in dry CH₂Cl₂ (37 mL) cooled at 0 °C were added NaHCO₃ (0.94 g, 11 mmol) and m-chloroperoxybenzoic acid (2.1 g, 12 mmol). After being stirred for 24 h at room temperature, the reaction mixture was diluted with sat. Na₂S₂O₃ aq (30 mL) at 0 °C, stirred for 30 min, and extracted with CH₂Cl₂ (20 mL × 3). The combined extracts were washed with brine, dried with Na₂SO₄, and concentrated. The crude material was purified with a SiO₂ column (50 g, hexane/EtOAc = 40/1 to 1/1) to give phenyltetrazole sulfone 10 (1.9 mg, 96%) as a light yellow oil. 10: R_f = 0.73 (19:1 toluene/ether); [α]_D²⁵ +0.22 (c 1.8, CHCl₃); IR (CHCl₃): 3008, 2957, 2931, 2884, 2859, 1731, 1713, 1596, 1497, 1463, 1341, 1255, 1153, 1076, 1030, 909, 839 cm^–1; ¹H NMR (600 MHz, CDCl₃): δ 7.68–7.58 (m, 5H), 7.34–7.28 (m, 5H), 4.50 (d, J = 12.0 Hz, 1H), 4.43 (d, J = 12.0 Hz, 1H), 4.09 (dd, J = 14.6, 2.0 Hz, 1H), 3.65 (dd, J = 6.4, 2.6 Hz, 1H), 3.48 (dd, J = 9.3, 5.6 Hz, 1H), 3.45 (dd, J = 14.6, 10.0 Hz, 1H), 3.36 (dd, J = 9.3, 6.1 Hz, 1H), 2.58 (dddq, J = 10.0, 2.7, 2.0, 6.4 Hz, 1H), 1.96 (dddq, J = 6.4, 6.1, 5.6, 6.4 Hz, 1H), 1.21 (d, J = 6.4 Hz, 3H), 0.98 (d, J = 6.4 Hz, 3H), 0.89 (s, 9H), 0.06 (s, 6H); ¹³C NMR (150 MHz, CDCl₃): δ 154.3, 138.6, 133.2, 131.6, 129.8 (2C), 128.5 (2C), 127.8 (2C), 127.6, 125.4 (2C), 77.8, 73.2, 72.3, 58.4, 38.7, 30.6, 26.2 (3C), 19.4, 18.5, 14.3, −4.0 (2C); HRMS (ESI) m/z: 567.2415 (calcd for C₂₇H₄₀N₄NaO₄SSi [M + Na]⁺, Δ −2.2 mmu).

Olefin 11a

To a stirred solution of phenyltetrazole sulfone 10 (1.9 g, 3.5 mmol) in dry DME (18 mL) cooled at −55 °C was added a 1.0 M solution of LHMDS in THF (3.5 mL, 3.5 mmol) dropwise under a nitrogen stream. The mixture was stirred at −55 °C for 30 min, then a solution of aldehyde 11 (0.39 g, 1.3 mmol) in DME (2 mL) was added dropwise for 10 min, and the resulting mixture was stirred at −55 °C for 2 h and allowed to warm to room temperature for 16 h. After the addition of brine (10 mL) at 0 °C, the reaction mixture was extracted with ether (10 mL × 3). The combined extracts were washed with brine, dried with Na₂SO₄, and concentrated. The crude material was purified with a SiO₂ column (50 g, hexane/acetone = 65/1 to 10/1) to give olefin 11a (0.82 g, 100%, E/Z = 1:2) and recovered sulfone 10 (0.94 g) as light yellow oils. 11a: R_f = 0.50 (4:1 hexane/ether); [α]_D²⁵ +21 (c 1.8, CHCl₃); IR (CHCl₃): 3006, 2975, 2960, 2858, 1454, 1364, 1254, 1210, 1096, 909, 838 cm^–1; ¹H NMR (600 MHz, CDCl₃): δ 7.37–7.30 (m, 10H), 5.79 [5.70] (dd, J = 11.0, 11.0 Hz, 1H), 5.48 [5.45] (dd, J = 11.0, 9.6 Hz, 1H), 4.94 [4.93] (d, J = 4.1 Hz, 1H), 4.57 (dd, J = 11.0, 4.7 Hz, 1H), 4.52–4.38 (m, 4H), 3.76 [3.70] (dd, J = 9.5, 6.4 Hz, 1H), 3.55 [3.44] (dd, J = 8.9, 5.6 Hz, 1H), 3.32–3.30 (m, 4H), 3.28 (dd, J = 8.3, 8.0 Hz, 1H), 2.65–2.62 [2.42] (m, 1H), 2.20 (dddq, J = 15.1, 7.0, 6.4, 7.0 Hz, 1H), 2.07 (ddq, J = 9.5, 5.2, 7.0 Hz, 1H), 1.98–1.96 (m, 1H), 1.71–1.63 (m, 2H), 1.10 [1.11] (d, J = 7.0 Hz, 3H), 1.00 [0.99] (d, J = 7.0 Hz, 3H), 0.97 (d, J = 7.0 Hz, 3H), 0.95 [0.94] (d, J = 7.0 Hz, 3H), 0.92 [0.91] (s, 9H), 0.06 [0.04] (s, 6H); ¹³C NMR (150 MHz, CDCl₃): δ 139.7, 139.0, 134.8 [136.1], 129.5 [129.4], 128.5 (2C), 128.2 (2C), 127.7, 127.6 (2C), 127.4 (2C), 127.2, 105.1 [104.9], 87.7 [87.5], 78.3 [76.8], 75.1, 73.0, 72.9, 70.6 [70.5], 55.1 [55.0], 46.3 [46.4], 42.2 [42.4], 39.1 [38.7], 36.0 [36.5], 35.3, 26.3 [26.2] (3C), 20.1 [20.0], 18.6, 14.9, 12.2, 10.0 [9.9], −3.6 [−3.8] (2C); chemical shifts of the E-isomer are within parentheses (square blankets); HRMS (ESI) m/z: 633.3931 (calcd for C₃₇H₅₈NaO₅Si [M + Na]⁺, Δ −2.0 mmu).

Diol 11b

A solution of olefin 11a (2.5 g, 4.1 mmol) in dry THF (14 mL) and i-PrOH (5.1 mL) was added in liquid ammonia (10 mL) at −78 °C. Calcium (0.49 g, 12 mmol) was added to the solution, and the mixture was stirred at −78 °C for 2 h. After the addition of ammonium chloride (4 g) and iron(III) nitrate nonahydrate (0.80 g), the mixture was stirred at −78 °C for 1 h and allowed to warm to room temperature for 5 h. The reaction mixture was diluted with water (20 mL) and extracted with ether (10 mL × 3). The combined extracts were washed with brine, dried with Na₂SO₄, and concentrated. The crude material was purified with a SiO₂ column (36 g, hexane/acetone = 65/1 to 5/1) to give diol 11b (1.2 g, 69%, E/Z = 1:2) as a colorless oil. 11b: R_f = 0.18 (1:2 hexane/EtOAc); [α]_D²⁵ +27 (c 1.3, CHCl₃); IR (CHCl₃): 3619, 3486, 3007, 2960, 2932, 2904, 2884, 2856, 2773, 1470, 1462, 1383, 1362, 1255, 1098, 1026, 837 cm^–1; ¹H NMR (600 MHz, CDCl₃): δ 5.66 [5.61] (dd, J = 11.0, 11.0 Hz, 1H), 5.52 [5.49] (dd, J = 11.0, 8.9 Hz, 1H), 4.92 [4.93] (d, J = 4.9 Hz, 1H), 4.63 [4.45] (dd, J = 18.0, 7.8 Hz, 1H), 3.64–3.52 (m, 3H), 3.33 [3.39] (m, 1H), 3.33 [3.32] (s, 3H), 2.81–2.74 (m, 1H), 2.56 (br s, 1H), 2.48 (br s, 1H), 2.26 (dddq, J = 13.9, 10.3, 3.4, 7.1 Hz, 1H), 2.06 (dddq, J = 12.7, 7.8, 7.0, 6.5 Hz, 1H), 1.88–1.77 (m, 2H), 1.61 (ddd, J = 12.8, 5.1, 2.3 Hz, 1H), 1.06 [1.05] (d, J = 6.5 Hz, 3H), 1.03 [1.02] (d, J = 6.5 Hz, 3H), 0.94 [0.92] (d, J = 6.5 Hz, 3H), 0.89 [0.84] (m, 3H), 0.89 [0.88] (s, 9H), 0.04 (s, 6H); ¹³C NMR (150 MHz, CDCl₃): δ 135.0 [135.7], 129.6 [129.3], 105.2 [105.1], 89.0, 80.5, 69.6 [69.3], 65.7, 55.4, 43.8 [43.7], 41.9, 40.3, 39.6, 36.7, 35.8 [35.7], 26.2 (3C), 19.6, 18.8, 15.6, 11.9, −3.6 [−4.0] (2C); chemical shifts of the E-isomer are within parentheses (square blankets); HRMS (ESI) m/z: 453.3001 (calcd for C₂₃H₄₆NaO₅Si [M + Na]⁺, Δ −1.1 mmu).

TBDPS Ether 12

To a stirred solution of diol 11b (1.0 g, 2.3 mmol) in dry DMF (12 mL) cooled at 0 °C were added imidazole (0.32 g, 4.6 mmol) and tert-butylchlorodiphenylsilane (0.60 mL, 2.3 mmol). After being stirred for 3 h at room temperature, the reaction mixture was diluted with water (20 mL) and extracted with EtOAc (10 mL × 3). The combined extracts were washed with water and brine, dried with Na₂SO₄, and concentrated. The crude material was purified with a SiO₂ column (34 g, hexane/EtOAc = 5/1 to 4/1) to give TBDPS ether 12 (1.5 g, 99%, E/Z = 1:2) as a colorless oil. 12: R_f = 0.69 (1:2 hexane/EtOAc); [α]_D²⁵ +24 (c 1.6, CHCl₃); IR (CHCl₃): 3671, 3491, 3009, 2960, 2932, 2896, 2858, 1712, 1656, 1589, 1471, 1428, 1362, 1333, 1254, 1222, 1111, 1027, 909, 734 cm^–1; ¹H NMR (600 MHz, CDCl₃): δ 7.72–7.70 (m, 4H), 7.42–7.40 (m, 6H), 5.69 [5.53] (dd, J = 10.2, 10.2 Hz, 1H), 5.46 [5.49] (dd, J = 10.2, 7.0 Hz, 1H), 4.92 [4.91] (d, J = 5.0 Hz, 1H), 4.50–4.56 (m, 1H), 3.73 (dd, J = 5.7, 2.3 Hz, 1H), 3.69 (dd, J = 10.0, 6.4 Hz, 1H), 3.60–3.56 (m, 2H), 3.40 (dd, J = 9.9, 8.0 Hz, 1H), 3.12 [3.34] (s, 3H), 2.68–2.66 (m, 1H), 2.25 [2.35] (dddq, J = 14.7, 4.3, 3.8, 7.0 Hz, 1H), 2.09–2.04 (m, 1H), 1.83–1.76 (m, 2H), 1.61 (m, 1H), 1.21 [1.20] (d, J = 7.0 Hz, 3H), 1.05 [1.07] (s, 9H), 1.01 [1.00] (d, J = 7.0 Hz, 3H), 0.98 [0.97] (d, J = 7.0 Hz, 3H), 0.89 (m, 3H), 0.84 [0.82] (s, 9H), −0.02 (s, 6H); ¹³C NMR (150 MHz, CDCl₃): δ 135.8 (2C), 135.4 [135.3], 135.0 (2C), 129.8 [129.7], 127.9 (4C), 127.8 (4C), 105.2, 89.4 [89.5], 77.7 [75.8], 69.9 [69.8], 66.8 [66.5], 55.4, 43.7 [43.5], 41.8 [41.5], 40.0, 35.8, 35.1, 27.1 (3C), 26.3 (3C), 20.5, 19.5, 18.6, 13.9, 12.4, 11.4, −3.9 (2C); chemical shifts of the E-isomer are within parentheses (square blankets); HRMS (ESI) m/z: 691.4185 (calcd for C₃₉H₆₄NaO₅Si₂ [M + Na]⁺, Δ −0.5 mmu).

Secondary Alcohol 12a

A mixture of TBDPS ether 12 (0.75 g, 1.1 mmol), NaHCO₃ (190 mg, 2.2 mmol), and palladium hydroxide 20% on carbon (0.19 g, 0.22 mmol) in ethanol (11 mL) was stirred under a hydrogen atmosphere at room temperature for 72 h. The mixture was filtered through a pad of Celite, and the residue was washed with EtOAc. The filtrate and the washings were combined and concentrated. The crude material was purified with a SiO₂ column (15 g, hexane/EtOAc = 20/1 to 5:1) to give secondary alcohol 12a (0.61 g, 82%) as a colorless oil. 12a: R_f = 0.54 (1:1 hexane/ether); [α]_D²⁵ +23 (c 1.7, CHCl₃); IR (CHCl₃): 3510, 2960, 2932, 2858, 1733, 1715, 1589, 1462, 1428, 1384, 1254, 1222, 1110, 1029, 909, 837, 704 cm^–1; ¹H NMR (600 MHz, CDCl₃): δ 7.67–7.65 (m, 4H), 7.41–7.38 (m, 6H), 4.92–4.91 (m, 1H), 3.85–3.80 (m, 1H), 3.70 (dd, J = 5.7, 2.2 Hz, 1H), 3.57 (dd, J = 8.0, 6.9 Hz, 1H), 3.52 (dd, J = 9.8, 7.7 Hz, 1H), 3.42–3.35 (m, 4H), 2.80 (br s, 1H), 2.28 (m, 1H), 2.09–2.04 (m, 1H), 2.09 (dtq, J = 12.8, 7.0, 7.0 Hz, 1H), 1.95–1.93 (m, 1H), 1.85–1.84 (m, 1H), 1.69–1.34 (m, 5H), 1.06–1.05 (m, 3H), 1.06 (s, 9H), 0.97 (d, J = 7.0 Hz, 3H), 0.96 (d, J = 7.0 Hz, 3H), 0.87–0.75 (m, 3H), 0.79 (s, 9H), −0.02 (s, 6H); ¹³C NMR (150 MHz, CDCl₃): δ 135.8 (2C), 129.6 (2C), 128.5 (4C), 127.7 (4C), 105.1, 90.3, 79.0, 73.0, 66.6, 55.4, 41.9, 39.6, 37.7, 35.1, 32.2, 29.0, 27.1 (3C), 26.3 (3C), 19.4, 18.4, 18.1, 16.9, 16.2, 15.5, 11.7, −3.7 (2C); HRMS (ESI) m/z: 693.4324 (calcd for C₃₉H₆₆NaO₅Si₂ [M + Na]⁺, Δ −1.2 mmu).

DMAla Ester 12b

To a solution of secondary alcohol 12a (597 mg, 0.890 mmol), N,N-dimethyl-l-alanine (208 mg, 1.78 mmol), and N,N-dimethyl-4-aminopyridine (DMAP) (260 mg, 2.13 mmol) in dry CH₂Cl₂ (6 mL) was added 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl) (374 mg, 1.95 mmol). After being stirred at room temperature for 12 h, the resulting mixture was diluted with water (5 mL) and extracted with CH₂Cl₂ (2 mL × 4). The combined extracts were washed with brine, dried with Na₂SO₄, and concentrated. The crude material was purified with a SiO₂ column (10 g, hexane/EtOAc = 1:0, 5:1 to 1:2) to give DMAla ester 12b (676 mg, 99%, S/R = 10:1 at C29 DMAla moiety) as a colorless oil. 12b: R_f = 0.41 (1:2 hexane/EtOAc); [α]_D²⁵ +9.3 (c 1.8, CHCl₃); IR (CHCl₃): 3072, 2959, 2932, 2858, 1724, 1471, 1384, 1254, 1110, 1030, 909, 837 cm^–1; ¹H NMR (600 MHz, CDCl₃): δ 7.65–7.62 (m, 4H), 7.42–7.38 (m, 6H), 5.35–5.32 (m, 1H), 4.91 [4.89] (d, J = 4.9 Hz, 1H), 3.76 [3.65] (dd, J = 10.0, 5.2 Hz, 1H), 3.42 (dd, J = 5.6, 3.3 Hz, 1H), 3.40–3.20 (m, 3H), 3.30 [3.28] (s, 3H), 2.35 [2.34] (s, 6H), 2.22 (dddq, J = 10.0, 7.9, 3.5, 6.7 Hz, 1H), 2.06 (ddq, J = 12.7, 6.7, 6.7 Hz, 1H), 1.89–1.81 (m, 1H), 1.65–1.60 (m, 2H), 1.63 (dddq, J = 10.0, 4.9, 2.0, 6.7 Hz, 1H), 1.60–1.54 (m, 4H), 1.29 [1.27] (d, J = 6.7 Hz, 3H), 1.08 [1.07] (d, J = 6.7 Hz, 3H), 1.05 (s, 9H), 0.92 [0.88] (d, J = 6.7 Hz, 3H), 0.91 [0.80] (d, J = 6.7 Hz, 3H), 0.84–0.75 (m, 3H), 0.80 (s, 9H), −0.04 [0.02] (s, 6H); ¹³C NMR (150 MHz, CDCl₃): δ 172.8, 135.8 (2C), 129.6 (2C), 128.5 (4C), 127.7 (4C), 104.9, 87.2, 78.6, 74.8 [74.6], 66.8, 63.2 [63.1], 54.8, 43.2 [43.1], 42.4, 41.8 (2C), 40.0, 37.1, 35.9, 30.6, 27.1 (3C), 26.2 [26.3] (3C), 20.0, 19.4, 18.4, 17.2, 16.6, 15.9, 15.1, 9.7, −3.8 (2C); chemical shifts of the minor diastereomers are within parentheses (square blankets); HRMS (ESI) m/z: 792.5034 (calcd for C₄₄H₇₅NNaO₆Si₂ [M + Na]⁺, Δ +0.3 mmu).

Hemiacetal 12c

A mixture of lactone 12b (38 mg, 50 μmol) in DME (6.2 mL) and 1 M HCl aq (2.5 mL) was stirred for 3 h at room temperature. The resulting mixture was neutralized with sat. NaHCO₃ aq (5 mL) at 0 °C and extracted with EtOAc (10 mL × 3). The combined extracts were washed with brine, dried with Na₂SO₄, and concentrated. The crude material was purified with a SiO₂ column (1 g, hexane/acetone = 10/1 to 1/1) to give hemiacetal 12c (31 mg, 84%, S/R = 3:1 at C29 DMAla moiety, 2.0:1 diastereomer mixture at C34) as a colorless oil. 12c: R_f = 0.15 (EtOAc); [α]_D²⁵ −3.3 (c 2.0, CHCl₃); IR (CHCl₃): 3598, 2959, 2932, 2859, 1721, 1462, 1384, 1254, 1110, 1043, 909, 837 cm^–1; ¹H NMR (600 MHz, CDCl₃): δ 7.66–7.63 (m, 4H), 7.42–7.40 (m, 6H), 5.44–5.43 (m, 1H), 5.06 [5.21] (d, J = 4.9 Hz, 1H), 3.78 [3.68] (dd, J = 5.2, 2.3 Hz, 1H), 3.57 [3.51] (dd, J = 8.1, 8.0 Hz, 1H), 3.41–3.34 (m, 2H), 3.29 [3.21] (q, J = 7.0 Hz, 1H), 2.33 [2.37] (s, 6H), 2.24 (dddq, J = 13.1, 4.9, 3.2, 7.0 Hz, 1H), 1.98 (dtq, J = 10.0, 5.2, 7.0 Hz, 1H), 1.96–1.52 (m, 9H), 1.22 [1.29] (d, J = 7.0 Hz, 3H), 1.19 (s, 9H), 1.16 (d, J = 7.0 Hz, 3H), 1.08 [1.06] (d, J = 7.0 Hz, 3H), 0.97 (d, J = 7.0 Hz, 3H), 0.94 (d, J = 7.0 Hz, 3H), 0.78 (s, 9H), −0.04 [0.02] (s, 6H); ¹³C NMR (150 MHz, CDCl₃): δ 172.9, 135.8 (2C), 129.7 (2C), 128.5 (4C), 127.7 (4C), 98.3 [98.2], 87.9, 78.8, 76.2, 66.6 [66.7], 62.7, 43.1, 42.9, 41.5 (2C), 36.8 [36.6], 36.1 [36.2], 28.4, 27.1 (3C), 26.2 (3C), 19.6, 19.4, 19.0, 18.4, 17.2, 15.2 [15.7], 12.5, 12.3, 10.2, −4.0 [−3.7] (2C); chemical shifts of the minor diastereomers are within parentheses (square blankets); HRMS (ESI) m/z: 778.4854 (calcd for C₄₃H₇₃NNaO₆Si₂ [M + Na]⁺, Δ −2.0 mmu).

Diol 13

To a stirred solution of hemiacetal 12c (27 mg, 36 μmol) in dry MeOH (2.3 mL) cooled at 0 °C was added sodium borohydride (8.1 mg, 0.214 mmol). After being stirred at 0 °C for 30 min, the resulting mixture was quenched with acetone (1 mL) and diluted with sat. NH₄Cl aq (3 mL) and extracted with EtOAc (2 mL × 3). The combined extracts were washed with brine, dried with Na₂SO₄, and concentrated. The crude material was purified with two SiO₂ columns (FL60D 0.7 g, hexane/EtOAc = 2:1 to 1:2; FL60D 0.7 g, CHCl₃/acetone = 5:1 to 1:1) to give diol 13 (25 mg, 91%, S/R = 2.0:1 at C29 DMAla moiety) as a colorless oil. None of the stereoisomers of the C29 DMAla ester after 13 were separable by SiO₂ column chromatography, and thus they were all used as mixtures. 13: R_f = 0.28 (1:1 CHCl₃/acetone); [α]_D²³ −7.2 (c 0.47, MeOH); IR (CHCl₃): 3392, 2958, 3052, 2931, 2858, 1714, 1472, 1462, 1428, 1255, 1185, 1109, 1055, 837 cm^–1; ¹H NMR (400 MHz, CDCl₃): δ 7.66–7.63 (m, 4H), 7.43–7.36 (m, 6H), 5.20 (m, 1H), 3.76 (dd, J = 9.9, 5.3 Hz, 1H), 3.70 (m, 1H), 3.54 (dd, J = 11.3, 6.4 Hz, 1H), 3.42–3.36 (m, 2H), 3.29 (q, J = 7.2 Hz, 1H), 3.05 (m, 1H), 2.37 [2.35] (s, 6H), 2.18 [2.17] (s, 1H), 2.08 [2.04] (s, 1H), 1.88–1.78 (m, 4H), 1.67–1.45 (m, 6H), 1.29 [1.25] (d, J = 7.2 Hz, 3H), 1.05 (s, 9H), 1.02 [1.00] (d, J = 7.1 Hz, 3H), 0.92 (d, J = 6.9 Hz, 3H), 0.86 (d, J = 7.6 Hz, 3H), 0.81 (d, J = 6.9 Hz, 3H), 0.78 [0.85] (s, 9H), −0.03 [0.02] (s, 3H), −0.13 [−0.04] (s, 3H); ¹³C NMR (100 MHz, CDCl₃): δ 174.9 [174.8], 135.6 (2C), 135.6 (2C), 134.0 (2C), 129.5 [129.6] (2C), 127.6 (4C), 78.4, 75.9 [76.0], 75.5 [75.3], 66.5 [67.1], 62.9 [62.8], 59.1 [59.2], 41.6 [41.5] (2C), 40.0 [39.6], 39.9 [38.5], 36.6 [38.4], 32.4 [32.6], 31.7 [31.8], 30.9 [30.7], 28.2 [29.2], 26.9 (3C), 26.0 [26.1] (3C), 19.2, 18.2 [18.4], 17.4, 17.4 [16.5], 14.7 [14.2], 14.5 [11.6], 9.9, −3.9 [−3.8], −4.2; chemical shifts of the minor diastereomers are within parentheses (square blankets); HRMS (ESI) m/z: 758.5210 (calcd for C₄₃H₇₆NO₆Si₂ [M + H]⁺, Δ −0.2 mmu).

Trityl Ether 13a

To a stirred solution of diol 13 (91 mg, 0.12 mmol) in dry CH₂Cl₂ (1.2 mL) was added trityl chloride (67 mg, 0.24 mmol), triethylamine (33 μL, 0.12 mmol), and DMAP (1.4 mg, 12 μmol) at room temperature. After being stirred for 15 h at room temperature, the resulting mixture was quenched with sat. NaHCO₃ aq (1.5 mL), and extracted with CH₂Cl₂ (2 mL × 3). The combined extracts were washed with brine, dried with Na₂SO₄, and concentrated. The crude material was purified with a SiO₂ column (4 g, hexane/EtOAc = 15:1 to 3:1) to give trityl ether 13a (107 mg, 90%, S/R = 2.0:1 at C29 DMAla moiety) as a colorless oil. 13a: R_f = 0.70 (1:1 CHCl₃/acetone); [α]_D²³ +10 (c 0.53, MeOH); IR (CHCl₃): 3070, 3008, 2958, 2879, 2858, 1715, 1472, 1462, 1449, 1255, 1219, 1184, 1110, 1089, 1073, 1033, 837 cm^–1; ¹H NMR (400 MHz, CDCl₃): δ 7.66–7.63 (m, 4H), 7.43 (d, J = 7.3 Hz, 6H), 7.41–7.34 (m, 6H), 7.30–7.14 (m, 9H), 5.21 (m, 1H), 3.77 [3.69] (dd, J = 10.1, 5.2 Hz, 1H), 3.43 (m, 1H), 3.39 [3.52] (dd, J = 10.1, 7.1 Hz, 1H), 3.28–3.21 (m, 2H), 3.02–2.96 (m, 2H), 2.34 [2.32] (s, 6H), 2.05 [2.17] (s, 1H), 1.89–1.76 (m, 4H), 1.60–1.39 (m, 6H), 1.27 [1.23] (d, J = 7.1 Hz, 3H), 1.06 (s, 9H), 0.94 (d, J = 6.8 Hz, 3H), 0.89 (d, J = 6.8 Hz, 3H), 0.87 (d, J = 6.9 Hz, 3H), 0.82 (d, J = 6.5 Hz, 3H), 0.78 [0.82] (s, 9H), −0.02 [0.03] (s, 3H), −0.12 [−0.03] (s, 3H); ¹³C NMR (100 MHz, CDCl₃): δ 174.7 [174.5], 144.5 (3C), 135.6 (2C), 135.6 (2C), 134.0 [133.9] (2C), 129.5 [129.6] (2C), 128.7 (6C), 127.7 (6C), 127.6 (4C), 126.8 (3C), 86.4, 78.5, 76.6 [75.3], 75.4 [75.1], 66.5 [67.2], 62.9 [62.8], 61.7, 41.6 (2C), 40.0, 39.7 [38.5], 36.7 [38.4], 31.0 [31.1], 30.9 [30.6], 29.0 [29.1], 28.2, 26.9 (3C), 26.0 [26.1] (3C), 19.3 [19.2], 18.2 [18.4], 17.7, 17.4 [16.5], 14.8 [14.4], 14.8 [11.6], 9.9, −3.9 [−3.7], −4.2; chemical shifts of the minor diastereomers are within parentheses (square blankets); HRMS (ESI) m/z: 1022.6122 (calcd for C₆₂H₈₉NNaO₆Si₂ [M + Na]⁺, Δ −0.4 mmu).

Acetate 14

A mixture of trityl ether 13a (99 mg, 98 μmol) and DMAP (25 mg, 0.21 mmol) in dry pyridine (1.7 mL) and acetic anhydride (0.80 mL) was stirred at 40 °C for 7 h. The resulting mixture was azeotropically concentrated with toluene. The crude material was purified with a SiO₂ column (10 g, hexane/EtOAc = 10/1 to 1/3) to give acetate 14 (103 mg, quant., S/R = 2.0:1 at C29 DMAla moiety) as a colorless oil. 14: R_f = 0.52 (5:9 hexane/EtOAc); [α]_D²⁴ +1.1 (c 1.4, MeOH); IR (CHCl₃): 3070, 3033, 3008, 2957, 2858, 1727, 1665, 1490, 1471, 1462, 1428, 1375, 1251, 1219, 1154, 1110, 1074, 1033, 967, 836 cm^–1; ¹H NMR (400 MHz, CDCl₃): δ 7.66–7.64 (m, 4H), 7.43 (d, J = 7.3 Hz, 6H), 7.41–7.34 (m, 6H), 7.30–7.17 (m, 9H), 4.92 (m, 1H), 4.76 (dd, J = 10.1, 2.5 Hz, 1H), 3.79 [3.66] (dd, J = 10.0, 5.2 Hz, 1H), 3.43–3.37 (m, 2H), 3.24–3.19 (m, 2H), 2.99 [3.48] (q, J = 7.1 Hz, 1H), 2.37 [2.36] (s, 6H), 1.99 [2.05] (s, 3H), 1.93–1.81 (m, 4H), 1.56–1.40 (m, 6H), 1.30 [1.27] (d, J = 7.1 Hz, 3H), 1.06 (s, 9H), 0.99 (d, J = 6.9 Hz, 3H), 0.95 (d, J = 6.8 Hz, 3H), 0.86 (d, J = 6.9 Hz, 3H), 0.80 [0.87] (s, 9H), 0.69 (d, J = 6.8 Hz, 3H), 0.01 [0.05] (s, 3H), −0.11 [−0.02] (s, 3H); ¹³C NMR (100 MHz, CDCl₃): δ 172.4, 170.6, 144.3 (3C), 135.6 (4C), 134.0 (2C), 129.5 (2C), 128.6 (6C), 127.7 (6C), 127.6 (4C), 126.9 (3C), 86.4, 78.7, 77.5 [75.2], 72.4 [72.3], 66.4 [67.1], 62.8, 61.2, 41.5 (2C), 39.8 [38.3], 36.5 [38.0], 35.9 [35.5], 30.8, 29.8, 29.5 [28.6], 27.5, 26.9 (3C), 26.0 [26.1] (3C), 20.9, 19.2, 18.3 [18.4], 17.0, 16.9, 15.5 [15.9], 15.0 [11.1], 9.7 [9.6], −3.9 [−3.7], −4.1; chemical shifts of the minor diastereomers are within parentheses (square blankets); HRMS (ESI) m/z: 1042.6406 (calcd for C₆₄H₉₂NO₇Si₂ [M + H]⁺, Δ −0.6 mmu).

Primary Alcohol 14a

To a stirred solution of acetate 14 (28.4 mg, 27.2 μmol) in dry ether (1 ml) was added formic acid (0.7 mL). After being stirred for 1 h at 40 °C, the resulting mixture cooled at 0 °C was quenched with sat. NaHCO₃ aq (5 mL) and extracted with ether (4 mL × 2). The combined extracts were washed with sat. NaHCO₃ aq and brine, dried with Na₂SO₄, and concentrated. The crude material containing primary alcohol and formate was dissolved in MeOH (1.5 mL) and 25% aq NH₃ (0.2 mL). After stirring at room temperature for 20 min, the resulting mixture cooled at 0 °C was diluted with brine (5 mL) and extracted with ether (3 mL × 3). The combined extracts were washed with brine, dried with Na₂SO₄, and concentrated. The crude material was purified with a SiO₂ column (5 g, hexane/EtOAc = 15/1 to 1/1) to give primary alcohol 14a (20.8 mg, 96%, S/R = 2.0:1 at C29 DMAla moiety) as a colorless oil. 14a: R_f = 0.40 (1:1 CHCl₃/acetone); [α]_D²⁵ +6.9 (c 0.90, CHCl₃); IR (CHCl₃): 3462, 2958, 2932, 2885, 2858, 1728, 1471, 1386, 1251, 1110, 1051, 837, 704 cm^–1; ¹H NMR (400 MHz, CDCl₃): δ 7.66–7.63 (m, 4H), 7.42–7.34 (m, 6H), 4.97 (br t, J = 7.1 Hz, 1H), 4.76 (dd, J = 9.9, 2.7 Hz, 1H), 3.78 (dd, J = 9.8, 5.2 Hz, 1H), 3.74 (m, 1 H), 3.64 (m, 1H), 3.42–3.36 (m, 2H), 3.26 [3.48] (q, J = 7.1 Hz, 1H), 2.39 (s, 6H), 2.04 [2.03] (s, 3H), 2.00–1.87 (m, 2H), 1.74–1.66 (m, 2H), 1.56–1.46 (m, 4H), 1.43–1.35 (m, 2H), 1.31 (d, J = 7.1 Hz, 3H), 1.26 (s, 1H), 1.06 (s, 9H), 0.95 (d, J = 6.8 Hz, 3H), 0.94 (d, J = 6.8 Hz, 3H), 0.92 (d, J = 6.9 Hz, 3H), 0.84 (d, J = 6.5 Hz, 3H), 0.79 [0.86] (s, 9H), 0.04 [−0.02] (s, 3H), −0.12 [−0.05] (s, 3H); chemical shifts of the minor diastereomers are within parentheses (square blankets); ¹³C NMR (100 MHz, CDCl₃): δ 172.3, 170.8, 135.8 (4C), 134.1 (2C), 129.5 (2C), 127.7 (4C), 78.7, 77.6, 72.5, 66.5, 62.8 [62.6], 60.6, 41.3 (2C), 39.8, 36.5, 36.0, 32.9 [32.7], 31.0 [30.9], 29.8 [29.7], 27.4, 26.9 (3C), 26.0 [26.2] (3C), 21.0, 19.3, 18.3 [18.5], 17.0, 16.8, 15.4, 14.9, 9.8, −3.9, −4.2; HRMS (ESI) m/z: 800.5329 (calcd for C₄₅H₇₈NO₇Si₂ [M + H]⁺, Δ +1.2 mmu).

Aldehyde 15

To a stirred solution of primary alcohol 14a (40 mg, 50 μmol) in dry CH₂Cl₂ (0.5 mL) were added dry pyridine (0.12 mL) and Dess–Martin periodinane (25 mg, 59 μmol).(21) After being stirred at room temperature for 2 h, the resulting mixture was diluted with a 1:1 mixture of sat. Na₂S₂O₃ aq and sat. NaHCO₃ aq (2 mL) at 0 °C and extracted with ether (2 mL × 4). The combined extracts were washed with brine, dried with Na₂SO₄, and concentrated. The crude material was purified with a SiO₂ column (FL60D 5 g, hexane/EtOAc = 2/1 to 1/2) to give aldehyde 15 (41 mg, quant., S/R = 2.0:1 at C29 DMAla moiety) as a colorless oil. 15: R_f = 0.68 (1:1 CHCl₃/acetone); [α]_D²⁵ +4.9 (c 0.73, CHCl₃); IR (CHCl₃): 2959, 2931, 2858, 1732, 1689, 1656, 1472, 1247, 1110, 1078, 669 cm^–1; ¹H NMR (400 MHz, CDCl₃): δ 9.77 (br s, 1H), 7.66–7.63 (m, 4H), 7.43–7.26 (m, 6H), 4.97 (br t, J = 7.1 Hz, 1H), 4.78 (dd, J = 9.5, 2.9 Hz, 1H), 3.77 [3.66] (dd, J = 9.9, 5.3 Hz, 1H), 3.42–3.36 (m, 2H), 3.28 (q, d = 7.1 Hz, 1H), 2.50–2.37 (m, 2H), 2.40 (s, 6H), 2.27 (m, 1H), 2.03 (s, 3H), 1.94–1.79 (m, 2H), 1.56–1.26 (m, 5H), 1.31 (d, J = 7.1 Hz, 3H), 1.05 (s, 9H), 1.01–0.96 (m, 9H), 0.84 (d, J = 7.2 Hz, 3H), 0.79 [0.86] (s, 9H), −0.02 [0.11] (s, 3H), −0.12 [−0.05] (s, 3H); ¹³C NMR (100 MHz, CDCl₃): δ 201.5, 172.4 [172.5], 170.5 [170.4], 135.9 (4C), 134.0 (2C), 129.8 [129.9] (2C), 127.9 (4C), 78.9, 77.2, 72.2, 66.7, 62.8, 45.4, 41.8 (2C), 40.1, 36.5, 29.8, 29.6 [29.4], 27.7, 26.8 (3C), 26.3, 26.0 (3C), 20.8, 19.4, 18.3, 17.2, 15.8, 14.8, 14.3, 9.9, −4.2, −4.1; chemical shifts of the minor diastereomers are within parentheses (square blankets); HRMS (ESI) m/z: 798.5131 (calcd for C₄₅H₇₆NO₇Si₂ [M + H]⁺, Δ −2.9 mmu).

Enamide 15a

A solution of aldehyde 15 (16 mg, 20 μmol), N-methylformamide (0.3 mL, 5.1 mmol), hydroquinone (4.5 mg, 40 μmol), and pyridinium p-toluenesulfonate (10 mg, 40 μmol) in dry benzene (15 mL) was stirred at reflux for 17 h under a nitrogen stream with continuous removal of water using MS3A. The resulting mixture was diluted with sat. NaHCO₃ aq (5 mL) at 0 °C and extracted with EtOAc (3 mL × 4). The combined extracts were washed with brine, dried with Na₂SO₄, and concentrated. The crude material was purified with a SiO₂ column (FL60D 0.5 g, hexane/EtOAc = 3/1 to 1:1) to give enamide 15a (10 mg, 60%) as a colorless oil. 15a: R_f = 0.55 (1:1 CHCl₃/acetone); [α]_D²⁵ −20 (c 1.2, CHCl₃); IR (CHCl₃): 2958, 2931, 2858, 1728, 1673, 1462, 1375, 1249, 1109, 1046, 837, 705 cm^–1; ¹H NMR (400 MHz, CDCl₃): δ 8.27 [8.03]¹ (s, 1H), 7.65–7.62 (m, 4H), 7.42–7.34 (m, 6H), 6.48 [7.15]¹ (d, J = 14.0 Hz, 1H), 5.01–4.93 (m, 2H), 4.78 (dd, J = 2.4, 11.0 Hz, 1H), 3.77 (dd, J = 9.8, 4.8 Hz, 1H), 3.42–3.35 (m, 2H), 3.22 (q, J = 7.1 Hz, 1H), 2.98 [2.97]¹ (s, 3H), 2.55 (m, 1H), 2.37 (s, 6H), 2.06 [2.05]¹ [2.03]² (s, 3H), 1.89–1.77 (m, 3H), 1.53–1.41 (m, 4H), 1.30 [1.29]² (d, J = 7.1 Hz, 3H), 1.05 (s, 9H), 1.01 (d, J = 6.9 Hz, 3H), 0.98 (d, J = 6.9 Hz, 3H), 0.96 (d, J = 6.8 Hz, 3H), 0.95 (d, J = 6.8 Hz, 3H), 0.77 [0.84]² (s, 9H), −0.05 (s, 3H), −0.14 (s, 3H); ¹³C NMR (100 MHz, CDCl₃): δ 172.3, 170.5, 162.0 [160.8]¹, 135.6 (4C), 133.9 (2C), 129.4 (2C), 129.3, 127.6 (4C), 110.4 [112.1]¹, 78.6, 72.1, 69.5, 66.4, 62.8, 41.4 (2C), 39.9, 36.9, 36.4, 32.9, 31.7, 30.1, 29.2 (3C), 27.5, 26.9 (3C), 20.9 [20.8]², 19.2, 18.2, 17.1, 15.4, 14.9, 11.1, 9.9, −3.9, −4.1; chemical shifts of the minor diastereomers are within parentheses as follows: []¹, 1.9:1 at C34 stereoisomers; []², S/R = 2.0:1 at C29 DMAla moiety; HRMS (ESI) m/z: 839.5408 (calcd for C₄₇H₇₉N₂O₇Si₂ [M + H]⁺, Δ −1.8 mmu).

C23–C34 Segment 16

Enamide 15a (22.1 mg, 26.3 μmol) was stirred for 32 h at 60 °C in a 0.6 M solution of ammonium fluoride in dry MeOH (2.6 mL, 1.56 mmol). The resulting mixture was quenched with sat. NaHCO₃ aq (2 mL) at 0 °C and extracted with EtOAc (4 mL × 2). The combined extracts were washed with brine, dried with Na₂SO₄, and concentrated. The crude material was purified with a SiO₂ column (FL60D, 2 g, CHCl₃/acetone = 5/1 to 1:1) to give C23–C34 segment 16 (12.1 mg, 76%) as a colorless oil. 16: R_f = 0.45 (1:1 CHCl₃/acetone); [α]_D²⁵ −29 (c 0.98, CHCl₃); IR (CHCl₃): 3020, 2958, 2932, 1733, 1656, 1457, 1375, 1249, 1077, 1044, 838, 670 cm^–1; ¹H NMR (400 MHz, CDCl₃): δ 8.28 [8.06]¹ (s, 1H), 6.48 [7.16]¹ (d, J = 14.0 Hz, 1H), 5.02–4.94 (m, 2H), 4.78 (dd, J = 10.0, 2.3 Hz, 1H), 3.58 (d, J = 5.3 Hz, 2H), 3.45 (m, 1H), 3.28 (q, J = 7.1 Hz, 1H), 3.01 [3.05]¹ (s, 3H), 2.58 (m, 1H), 2.39 [2.36]² (s, 6H), 2.07 [2.06]² (s, 3H), 1.88–1.75 (m, 2H), 1.66–1.46 (m, 4H), 1.40 (m, 1H), 1.31 (d, J = 7.1 Hz, 3H), 1.25 (s, 1H), 1.02 (d, J = 6.8 Hz, 3H), 0.94 (d, J = 6.9 Hz, 6H), 0.89 [0.88]² (s, 9H), 0.83 (d, J = 6.9 Hz, 3H), 0.10 [0.05]² (s, 3H), 0.03 [0.01]² (s, 3H); ¹³C NMR (100 MHz, CDCl₃): δ 172.5, 170.5, 162.2 [161.0]¹, 129.5, 110.5 [112.1]¹, 81.6 [81.7]¹, 76.2, 72.1 [72.2]¹, 66.1, 62.8, 41.4 [41.6]² (2C), 38.7, 36.9, 33.0, 31.8, 30.2, 29.3, 27.6, 26.0 (3C), 20.9, 19.4, 18.2, 16.6, 15.8, 15.3, 9.6, −4.1, −4.3; chemical shifts of the minor diastereomers are within parentheses as follows: []¹, 1.9:1 at C34 stereoisomers; []², S/R = 2.0:1 at C29 DMAla moiety; HRMS (ESI) m/z: 623.4066 (calcd for C₃₁H₆₀N₂NaO₇Si [M + Na]⁺, Δ −0.1 mmu).

Acetonide 17b

A solution of (+)-benzyl ether 17(22) (360 mg, 1.63 mmol) in dry THF (5.4 mL) and i-PrOH (2 mL) was added in liquid ammonia (4.1 mL) at −78 °C. Calcium (0.14 g, 3.6 mmol) was added to the solution, and the mixture was stirred at −78 °C for 1 h 20 min. After the addition of ammonium chloride (2 g) and iron(III)nitrate nonahydrate (0.25 g), the mixture was stirred at −78 °C for 1 h and allowed to warm to room temperature for 6 h. The reaction mixture was diluted with water (8 mL) and extracted with EtOAc (5 mL × 6). The combined extracts were washed with brine, dried with Na₂SO₄, and concentrated to give known diol 17a (0.25 g), which was used for the next step without further purification.

To a stirred solution of the above crude diol 17a (0.25 g) in dry CH₂Cl₂ (8.3 mL) and 2,2-dimethoxypropane (1.0 mL, 8.0 mmol) was added 10-camphorsulfonic acid (19 mg, 8.2 μmol). After stirring at room temperature for 2.5 h, the resulting mixture was quenched with sat. NaHCO₃ aq (2 mL) and extracted with CH₂Cl₂ (1 mL × 3). The combined extracts were washed with brine, dried with Na₂SO₄, and concentrated. The crude material was purified with a SiO₂ column (12 g, hexane/acetone = 30/1 to 10:1) to give acetonide 17b (280 mg, 99%) as a colorless oil. 17b: R_f = 0.82 (1:1 hexane/EtOAc); [α]_D²⁵ +20 (c 1.4, CHCl₃); IR (CHCl₃): 3078, 2998, 2960, 2934, 2912, 2864, 1641, 1459, 1385, 1165, 1107, 1061, 1008, 896, 822 cm^–1; ¹H NMR (400 MHz, CDCl₃): δ 5.90 (tdd, J = 6.9, 17.0, 7.2 Hz, 1H), 5.08 (dd, J = 17.0, 2.0 Hz, 1H), 5.04 (dd, J = 7.2, 2.0 Hz, 1H), 3.68 (dd, J = 11.6, 5.2 Hz, 1H), 3.55 (m, 1H), 3.50 (t, J = 11.6 Hz, 1H), 2.37 (m, 1H), 2.15 (m, 1H), 1.70 (m, 1H), 1.43 (s, 3H), 1.39 (s, 3H), 0.75 (d, J = 6.6 Hz, 3H); ¹³C NMR (100 MHz, CDCl₃): δ 134.9, 116.3, 98.2, 74.7, 65.9, 37.4, 33.4, 29.8, 19.1, 12.5; HRMS was not available due to the low detectable sensitivity on ESI-MS.

Aldehyde 17c

To a solution of acetonide 17b (430 mg, 2.53 mmol) in acetone–H₂O (3:1, 25 mL) were added N-methylmorpholine N-oxide (593 mg, 5.06 mmol) and a 0.1 M solution of osmium(VIII)oxide in tert-butyl alcohol (1.26 mL, 126 μmol) at 0 °C. After stirring at room temperature for 1 h, sodium periodate (1.46 g, 6.8 mmol) was added. After stirring at room temperature for 1 h, the resulting mixture was quenched with sat. Na₂S₂O₃ aq (10 mL) and extracted with ether (5 mL × 3). The combined extracts were washed with sat. Na₂S₂O₃ aq and brine, dried with Na₂SO₄, and concentrated. The crude material was purified with a SiO₂ column (25 g, pentane/ether = 5/1 to 1:1) to give aldehyde 17c (448 mg, quant.) as a colorless oil. 17c: R_f = 0.82 (1:1 hexane/EtOAc); [α]_D²⁵ +27 (c 1.8, CHCl₃); IR (CHCl₃): 3025, 2996, 2961, 2930, 2872, 2734, 1729, 1460, 1375, 1249, 1198, 1167, 1111, 1061, 1046 cm^–1; ¹H NMR (400 MHz, CDCl₃): δ 9.79 (dd, J = 2.8, 1.8 Hz, 1H), 4.05 (ddd, J = 9.2, 6.3, 3.5 Hz, 1H), 3.73 (dd, J = 11.5, 5.5 Hz, 1H), 3.56 (t, J = 11.5 Hz, 1H), 2.59 (ddd, J = 16.0, 6.3, 1.8 Hz, 1H), 2.51 (ddd, J = 16.0, 9.2, 2.8 Hz, 1H), 1.75 (m, 1H), 1.46 (s, 3H), 1.37 (s, 3H), 0.77 (d, J = 6.7 Hz, 3H); ¹³C NMR (100 MHz, CDCl₃): δ 201.5, 98.4, 71.1, 65.8, 46.8, 34.0, 29.5, 19.0, 12.4; HRMS (ESI) m/z: 195.0971 (calcd for C₉H₁₆NaO₃ [M + Na]⁺, Δ −2.6 mmu).

Conjugated Ester 18

To a stirred solution of triethyl 4-phosphonocrotonate (2.0 mL, 8.9 mmol) in dry THF (68 mL) cooled at −40 °C was added a 0.5 M solution of lithium diisopropylamide in THF (15 mL, 7.5 mmol) dropwise. The mixture was stirred at −40 °C for 25 min, then a solution of aldehyde 17c (436 mg, 2.53 mmol) in dry THF (13 mL) was added dropwise, and the resulting mixture was stirred at −40 °C for 20 min. The reaction was quenched by the addition of sat. NH₄Cl (20 mL) and extracted with ether (5 mL × 4). The combined extracts were washed with brine, dried with Na₂SO₄, and concentrated. The crude material was purified with a SiO₂ column (25 g, hexane/EtOAc = 10/1 to 1/1) to give conjugated ester 18 (579 mg, 82%, 4E/4Z = 10:1) as a colorless oil. 18: R_f = 0.64 (1:1 hexane/EtOAc); [α]_D²⁵ +21 (c 1.2, CHCl₃); IR (CHCl₃): 2996, 2960, 2860, 1703, 1644, 1618, 1462, 1370, 1266, 1210, 1060, 668 cm^–1; ¹H NMR (400 MHz, CDCl₃): δ 7.27 (ddd, J = 15.5, 7.4, 3.1 Hz, 1H), 6.21–6.20 (m, 2H), 5.78 (d, J = 15.5 Hz, 1H), 4.19 (q, J = 7.1 Hz, 2H), 3.67 (dd, J = 11.6, 5.2 Hz, 1H), 3.55 (ddd, J = 10.2, 7.3, 3.1 Hz, 1H), 3.49 (t, J = 11.6, 1H), 2.47 (m, 1H), 2.28 (m, 1H), 1.68 (m, 1H), 1.42 (s, 3H), 1.37 (s, 3H), 1.29 (t, J = 7.1 Hz, 3H), 0.75 (d, J = 6.7 Hz, 3H); ¹³C NMR (100 MHz, CDCl₃): δ 167.3, 144.9, 140.3, 130.1, 119.7, 98.4, 74.5, 65.9, 60.2, 36.6, 33.6, 29.8, 19.1, 14.4, 12.6; NMR data for 4E isomer are shown; HRMS (ESI) m/z: 291.1560 (calcd for C₁₅H₂₄NaO₄ [M + Na]⁺, Δ −1.2 mmu).

Diol 18a

A solution of conjugated ester 18 (390 mg, 1.45 mmol) and pyridinium p-toluenesulfonate (80 mg, 0.32 mmol) in EtOH (48 mL) was stirred at 35 °C for 2 days. The reaction was quenched by the addition of sat. NH₄Cl (50 mL) and extracted with EtOAc (20 mL × 3). The combined extracts were washed with brine, dried with Na₂SO₄, and concentrated. The crude material was purified with a SiO₂ column (10 g, hexane/EtOAc = 5/1 to 1/5) to give diol 18a (303 mg, 92%) as a colorless oil. 18a: R_f = 0.21 (5:9 hexane/EtOAc); [α]_D²⁵ +17 (c 0.85, CHCl₃); IR (CHCl₃): 3481, 3011, 2984, 2937, 2904, 1702, 1644, 1618, 1270, 1140, 1032, 1001, 670 cm^–1; ¹H NMR (400 MHz, CDCl₃): δ 7.27 (dd, J = 15.2, 10.8 Hz, 1H), 6.30–6.41 (m, 2H), 5.80 (d, J = 15.2 Hz, 1H), 4.18 (q, J = 7.1 Hz, 2H), 3.75 (dd, J = 10.8, 3.6 Hz, 1H), 3.68–3.60 (m, 2H), 3.21 (br s, 1H), 2.96 (br s, 1H), 2.51 (m, 1H), 2.32 (m, 1H), 1.75 (m, 1H), 1.28 (t, J = 7.1 Hz, 3H), 0.88 (d, J = 7.0 Hz, 3H); ¹³C NMR (100 MHz, CDCl₃): δ 167.2, 144.4, 139.8, 131.2, 120.2, 76.1, 67.6, 60.2, 39.6, 38.9, 14.2, 13.7; HRMS (ESI) m/z: 251.1232 (calcd for C₁₂H₂₀NaO₄ [M + Na]⁺, Δ −2.7 mmu).

TBDPS Ether 19

To a stirred solution of diol 18a (35.2 mg, 0.15 mmol) in dry DMF (0.8 mL) cooled at 0 °C were added imidazole (21 mg, 0.31 mmol) and tert-butylchlorodiphenylsilane (60 μL, 0.22 mmol). After being stirred for 8 h at room temperature, the reaction mixture was diluted with water (3 mL) and extracted with ether (2 mL × 3). The combined extracts were washed with brine, dried with Na₂SO₄, and concentrated. The crude material was purified with a SiO₂ column (FL60D 3 g, hexane/EtOAc = 5/1 to 1/1) to give TBDPS ether 19 (72 mg, quant.) as a colorless oil. 19: R_f = 0.85 (5:8 hexane/EtOAc); [α]_D²⁵ +15 (c 3.6, CHCl₃); IR (CHCl₃): 3470, 3009, 2961, 2931, 2860, 1703, 1643, 1618, 1471, 1370, 1268, 1002, 760 cm^–1; ¹H NMR (400 MHz, CDCl₃): δ 7.69–7.65 (m, 4H), 7.48–7.39 (m, 6H), 7.30 (ddd, J = 15.4, 6.7, 3.4 Hz, 1H), 6.31–6.18 (m, 2H), 5.80 (d, J = 15.4 Hz, 1H), 4.20 (q, J = 7.1 Hz, 2H), 3.82–3.72 (m, 2H), 3.63 (m, 1H), 2.86 (br s, 1H), 2.56–2.26 (m, 2H), 1.87 (m, 1H), 1.30 (t, J = 7.1 Hz, 3H), 1.06 (s, 9H), 0.82 (d, J = 6.9 Hz, 3H); ¹³C NMR (100 MHz, CDCl₃): δ 167.2, 144.8, 140.6, 135.6 (4C), 132.7 (2C), 130.5, 130.0 (2C), 127.8 (4C), 119.8, 75.6, 68.8, 60.1, 39.5, 38.5, 26.8 (3C), 19.1, 14.4, 13.4; HRMS (ESI) m/z: 489.2431 (calcd for C₂₈H₃₈NaO₄Si [M + Na]⁺, Δ +0.6 mmu).

MTM Ether 19a

A solution of TBDPS ether 19 (30 mg, 64 μmol) in dry DMSO (0.38 mL), acetic acid (48 μL, 0.84 mmol) and acetic anhydride (0.27 mL) was stirred at 40 °C for 16 h. The reaction mixture was quenched with sat. NaHCO₃ aq (10 mL) at 0 °C and extracted with ether (5 mL × 3). The combined extracts were washed with brine, dried with Na₂SO₄, and concentrated. The crude material was purified with a SiO₂ column (FL60D 3 g, hexane/ether = 25/1 to 5/1) to give MTM ether 19a (18 mg, 54%) as a colorless oil. 19a: R_f = 0.68 (1:1 hexane/ether); [α]_D²⁵ −10 (c 0.63, CHCl₃); IR (CHCl₃): 2960, 2930, 2859, 1703, 1643, 1618, 1428, 1303, 1266, 1001, 919, 704 cm^–1; ¹H NMR (400 MHz, CDCl₃): δ 7.67–7.64 (m, 4H), 7.45–7.36 (m, 6H), 7.25 (dd, J = 15.4, 10.1 Hz, 1H), 6.25–6.10 (m, 2H), 5.78 (d, J = 15.4 Hz, 1H), 4.58 (d, J = 11.5 Hz, 1H), 4.53 (d, J = 11.5 Hz, 1H), 4.20 (q, J = 7.1 Hz, 2H), 3.83 (m, 1H), 3.60 (d, J = 6.2 Hz, 2H), 2.38 (m, 1H), 2.28 (m, 1H), 2.05 (s, 3H), 1.98 (m, 1H), 1.30 (t, J = 7.1 Hz, 3H), 1.07 (s, 9H), 0.88 (d, J = 5.4 Hz, 3H); ¹³C NMR (100 MHz, CDCl₃): δ 167.3, 144.6, 140.8, 135.6 (4C), 133.7 (2C), 130.4, 129.7 (2C), 127.7 (4C), 119.7, 77.1, 73.7, 65.6, 60.3, 38.2, 33.8, 26.9 (3C), 19.3, 14.3, 14.1, 12.3; HRMS (ESI) m/z: 549.2470 (calcd for C₃₀H₄₂NaO₄SSi [M + Na]⁺, Δ −0.1 mmu).

Carboxylic Acid 20

A solution of MTM ether 19a (35 mg, 67 μmol) in MeOH (6 mL) and 4 M aq LiOH (0.76 mL, 3 mmol) was stirred at room temperature for 16 h. The reaction mixture was quenched with 1 M aq HCl (10 mL) at 0 °C and extracted with EtOAc (4 mL × 3). The combined extracts were washed with brine, dried with Na₂SO₄, and concentrated. The crude material was purified with a SiO₂ column (FL60D 2 g, hexane/EtOAc = 2/1 to 1/1) to give carboxylic acid 20 (33 mg, quant.) as a colorless oil. 20: R_f = 0.21 (1:1 hexane/ether); [α]_D²⁵ −13 (c 2.7, CHCl₃); IR (CHCl₃): 3004, 2978, 2933, 2874, 1698, 1636, 1617, 1418, 1384, 1111, 1075, 668 cm^–1; ¹H NMR (400 MHz, CDCl₃): δ 7.67–7.64 (m, 4H), 7.45–7.31 (m, 7H), 6.28–6.15 (m, 2H), 5.79 (d, J = 15.4 Hz, 1H), 4.58 (d, J = 11.5 Hz, 1H), 4.54 (d, J = 11.5 Hz, 1H), 3.85 (m, 1H), 3.61 (d, J = 6.0 Hz, 2H), 2.40 (m, 1H), 2.30 (m, 1H), 2.06 (s, 3H), 1.98 (m, 1H), 1.07 (s, 9H), 0.89 (d, J = 7.0 Hz, 3H), −COOH signal was not observed; ¹³C NMR (100 MHz, CDCl₃): δ 172.1, 147.1, 142.4, 135.7 (4C), 133.8 (2C), 130.2, 129.8 (2C), 127.7 (4C), 118.8, 77.2, 73.7, 65.7, 38.3, 33.8, 26.9 (3C), 19.3, 14.2, 12.2; HRMS (ESI) m/z: 521.2166 (calcd for C₂₈H₃₈NaO₄SSi [M + Na]⁺, Δ −0.9 mmu).

TBS Ether 19b

To a stirred solution of TBDPS ether 19 (390 mg, 0.84 mmol) in dry CH₂Cl₂ (2.8 mL) were added imidazole (114 mg, 1.67 mmol) and tert-butylchlorodimethylsilane (TBSCl) (189 mg, 1.25 mmol) at 0 °C. After being stirred for 16.5 h at room temperature, imidazole (57 mg, 0.84 mmol) and TBSCl (126 mg, 0.84 mmol) were added. After being stirred for 8.5 h at room temperature, imidazole (57 mg, 0.84 mmol) and TBSCl (126 mg, 0.84 mmol) were added again. After being stirred for 15 h at room temperature, the reaction mixture was diluted with water (3 mL) and extracted with ether (3 mL × 3). The combined extracts were washed with brine, dried with Na₂SO₄, and concentrated. The crude material was purified with a SiO₂ column (16 g, hexane/EtOAc = 20/1 to 1/1) to give TBS ether 19b (455 mg, 94%) as a colorless oil. 19b: R_f = 0.68 (4:1 hexane/EtOAc); [α]_D²⁵ −28 (c 0.99, CHCl₃); IR (CHCl₃): 3052, 2957, 2930, 2857, 1702, 1641, 1471, 1257, 1111, 835, 613 cm^–1; ¹H NMR (400 MHz, CDCl₃): δ 7.65–7.63 (m, 4H), 7.44–7.34 (m, 6H), 7.25 (dd, J = 15.6, 9.7 Hz, 1H), 6.18–5.97 (m, 2H), 5.77 (d, J = 15.6 Hz, 1H), 4.21 (q, J = 7.2 Hz, 2H), 3.85 (m, 1H), 3.62 (dd, J = 10.1, 7.2 Hz, 1H), 3.48 (dd, J = 10.1, 6.6 Hz, 1H), 2.32–2.17 (m, 2H), 1.92 (m, 1H), 1.30 (t, J = 7.2 Hz, 3H), 1.05 (s, 9H), 0.84 (s, 9H), 0.83 (d, J = 6.3 Hz, 3H), 0.02 (s, 2H), −0.01 (s, 2H), −0.03 (s, 2H); ¹³C NMR (100 MHz, CDCl₃): δ 167.3, 144.9, 141.6, 135.6 (4C), 133.8 (2C), 130.3, 129.6 (2C), 127.6 (4C), 119.5, 72.7, 66.0, 60.2, 41.3, 36.6, 26.9 (3C), 25.8 (3C), 19.2, 18.0, 14.3, 11.9, −4.3, −4.6; HRMS (ESI) m/z: 603.3301 (calcd for C₃₄H₅₂NaO₄Si₂ [M + Na]⁺, Δ +0.5 mmu).

Carboxylic Acid 21

Prepared from TBS ether 19b as in the case with carboxylic acid 20 in 89% yield. 21: R_f = 0.29 (1:1 hexane/ether); [α]_D²⁵ −29 (c 1.1, CHCl₃); IR (CHCl₃): 3072, 2957, 2930, 2857, 2665, 1687, 1638, 1616, 1427, 1258, 1110, 938, 853, 614 cm^–1; ¹H NMR (400 MHz, CDCl₃): δ 7.66–7.63 (m, 4H), 7.45–7.34 (m, 6H), 7.31 (m, 1H), 6.23–5.90 (m, 2H), 5.78 (d, J = 15.2 Hz, 1H), 3.87 (m, 1H), 3.62 (dd, J = 10.2, 7.2 Hz, 1H), 3.47 (dd, J = 10.2, 6.5 Hz, 1H), 2.35–2.20 (m, 2H), 1.92 (m, 1H), 1.06 (s, 9H), 0.84 (s, 9H), 0.83 (d, J = 6.4 Hz, 3H), −0.00 (s, 3H), −0.02 (s, 3H), −COOH signal was not observed; ¹³C NMR (100 MHz, CDCl₃): δ 172.6, 147.2, 143.2, 135.6 (4C), 133.8 (2C), 130.1, 129.6 (2C), 127.6 (4C), 118.5, 72.7, 65.9, 41.4, 36.7, 26.9 (3C), 25.8 (3C), 19.2, 18.0, 11.9, −4.3, −4.6; HRMS (ESI) m/z: 575.2983 (calcd for C₃₂H₄₈NaO₄Si₂ [M + Na]⁺, Δ ±0.0 mmu).

Conjugated Ester 22a

Prepared from aldehyde 22 as in the case with conjugated ester 18 in 89% yield (4E/4Z = 3.6:1). 4E isomer was partially purified by a SiO₂ column (FL60D, hexane/ether = 100/1 to 10/1). 22a: R_f = 0.54 (10:1 hexane/ether); IR (CHCl₃): 3027, 2955, 2929, 2857, 1701, 1642, 1617, 1471, 1257, 1097, 838 cm^–1; ¹H NMR (400 MHz, CDCl₃): δ 7.25 (dd, J = 15.4, 10.6 Hz, 1H), 6.26 (dd, J = 15.4, 10.6 Hz, 1H), 6.07 (dd, J = 15.4, 8.2 Hz, 1H), 5.80 (d, J = 15.4 Hz, 1H), 4.19 (q, J = 7.1 Hz, 2H), 3.68 (dd, J = 9.8, 6.0 Hz, 2H), 3.63 (dd, J = 9.8, 6.0 Hz, 2H), 2.45 (dtt, J = 9.8, 6.0, 6.0 Hz, 1H), 1.29 (t, J = 7.1 Hz, 3H), 0.88 (s, 18H), 0.03 (s, 12H); ¹³C NMR (100 MHz, CDCl₃): δ 167.3, 145.0, 142.7, 130.0, 120.0, 62.5 (2C), 60.2, 47.9, 25.9 (6C), 18.4 (2C), 14.4, −5.4 (4C); NMR data for 4E isomer are shown; HRMS (ESI) m/z: 451.2654 (calcd for C₂₂H₄₄NaO₄Si₂ [M + Na]⁺, Δ −1.7 mmu).

Carboxylic Acid 23

To a stirred solution of conjugated ester 22a (36 mg, 84 μmol) in THF–H₂O–MeOH (1:1:1, 0.75 mL) was added lithium hydroxide monohydrate (14 mg, 0.33 mmol). After stirring at room temperature for 24 h, the reaction mixture was neutralized with sat. NH₄Cl (5 mL) and extracted with EtOAc (3 mL × 3). The combined extracts were washed with brine, dried with Na₂SO₄, and concentrated. The crude material was purified with a SiO₂ column (FL60D 0.5 g, hexane/EtOAc = 5/1 to 1/1) to give carboxylic acid 23 (25 mg, 76%) as a colorless oil. 23: R_f = 0.50 (2:1 CHCl₃/acetone); IR (CHCl₃): 3035, 2955, 2929, 2857, 2650, 1687, 1638, 1616, 1471, 1256, 1110, 1003, 939, 835 cm^–1; ¹H NMR (400 MHz, CDCl₃): δ 7.92 (br s, 1H) 7.33 (dd, J = 15.3, 10.9 Hz, 1H), 6.30 (dd, J = 15.4, 10.9 Hz, 1H), 6.13 (dd, J = 15.4, 8.1 Hz, 1H), 5.84 (d, J = 15.3 Hz, 1H), 3.69 (dd, J = 9.5, 5.9 Hz, 2H), 3.64 (dd, J = 9.5, 5.9 Hz, 2H), 2.47 (m, 1H), 0.88 (s, 18H), 0.03 (s, 12H); ¹³C NMR (100 MHz, CDCl₃): δ 174.3, 147.2, 144.2, 129.8, 118.7, 62.5 (2C), 48.0, 25.9 (6C), 18.3 (2C), −5.4 (4C); HRMS (ESI) m/z: 423.2337 (calcd for C₂₀H₄₀NaO₄Si₂ [M + Na]⁺, Δ −2.0 mmu).

Ester 20a

To a stirred solution of carboxylic acid 20 (25 mg, 50 μmol) in dry THF (0.27 mL) were added a 0.4 M solution of triethylamine in THF (0.2 mL, 80 μmol) and a 0.3 M solution of 2,4,6-trichlorobenzoyl chloride in THF (0.2 mL, 60 μmol). After stirring at room temperature for 2.5 h, a solution of the C23–C34 segment 16 (12.1 mg, 20.1 μmol) in dry toluene (0.5 mL) and DMAP (7.3 mg, 60 μmol) were added at 0 °C. After stirring at room temperature for 30 min, the reaction mixture was quenched with sat. NaHCO₃ aq (2 mL) at 0 °C and extracted with ether (2 mL × 3). The combined extracts were washed with brine, dried with Na₂SO₄, and concentrated. The crude material was purified with a SiO₂ column (FL60D 3 g, CHCl₃/acetone = 20/1 to 1/1) to give ester 20a (14.2 mg, 66%) as a colorless oil. 20a: R_f = 0.68 (1:1 CHCl₃/acetone); [α]_D²⁵ −25 (c 0.46, CHCl₃); IR (CHCl₃): 3026, 2985, 2959, 2932, 2858, 1731, 1656, 1462, 1375, 1250, 1110, 1046, 772 cm^–1; ¹H NMR (400 MHz, CDCl₃): δ 8.28 [8.06]¹ (s, 1H), 7.66–7.63 (m, 4H), 7.43–7.36 (m, 6H), 7.23 (dd, J = 15.4, 10.2 Hz, 1H), 6.49 [7.16]¹ (d, J = 14.0 Hz, 1H), 6.25–6.12 (m, 2H), 5.78 (d, J = 15.4 Hz, 1H), 5.00–4.95 (m, 2H), 4.79 (dd, J = 10.0, 2.9 Hz, 1H), 4.57 (d, J = 11.6 Hz, 1H), 4.53 (d, J = 11.6 Hz, 1H), 4.32 (dd, J = 10.9, 5.0 Hz, 1H), 3.93 (dd, J = 10.9, 8.3 Hz, 1H), 3.83 (m, 1H), 3.60 (d, J = 6.0 Hz, 2H), 3.42 (t, J = 4.9 Hz, 1H), 3.22 (q, J = 7.2 Hz, 1H), 3.01 [3.05]¹ (s, 3H), 2.54 (m, 1H), 2.38 [2.35]² (s, 6H), 2.36–2.28 (m, 2H), 2.07 (s, 3H), 2.04 (s, 3H), 2.03–1.98 (m, 2H), 1.84 (m, 1H), 1.67–1.48 (m, 4H), 1.40 (m, 1H), 1.30 [1.26]² (d, J = 7.2 Hz, 3H), 1.06 (s, 9H), 1.03 (d, J = 6.9 Hz, 3H), 1.01 (d, J = 6.8 Hz, 3H), 0.96 (d, J = 6.8 Hz, 3H), 0.94 (d, J = 7.2 Hz, 3H), 0.90–0.88 (m, 3H), 0.90 [0.88]² (s, 9H), 0.03 (s, 6H); ¹³C NMR (100 MHz, CDCl₃): δ 172.5, 170.5, 167.3, 162.1 [160.9]¹, 144.6 [144.8]¹, 140.9 [141.0]¹, 135.6 (4C), 133.6 (2C), 130.3, 129.7 (2C), 129.3, 127.6 (4C), 119.6 [119.4]¹, 110.5 [112.1]¹, 78.8, 77.2, 77.1, 76.5, 73.7, 72.1, 65.7, 62.9, 41.5 [41.3]² (2C), 38.2, 37.1, 36.9, 36.1, 33.7, 33.0, 30.1, 29.7, 27.5, 26.9 (3C), 26.1 (3C), 20.9, 19.3, 18.3, 16.6, 15.7, 15.4, 14.1, 12.2, 11.4, 9.6, −3.8, −4.2; chemical shifts of the minor diastereomers are within parentheses as follows: []¹, 1.9:1 at C34 stereoisomers; []², S/R = 5.0:1 at C29 DMAla moiety; HRMS (ESI) m/z: 1081.6386 (calcd for C₅₉H₉₇N₂O₁₀SSi₂ [M + H]⁺, Δ −1.7 mmu).

Secondary Alcohol 24

To a stirred solution of ester 20a (5.6 mg, 5.2 μmol) in THF–H₂O (4:1, 0.47 mL) were added 2,6-lutidine (0.96 mL, 8.3 mmol) and silver(I)nitrate (130 mg, 0.78 mmol). After stirring at room temperature for 6 h in the dark, the reaction mixture was diluted with EtOAc (2 mL) and filtered through a pad of Celite, and the residue was washed with EtOAc. The combined filtrate and washings were with water and brine, dried with Na₂SO₄, and concentrated. The crude material was purified with a SiO₂ column (FL60D 0.5 g, CHCl₃/acetone = 20/1 to 5/1) to give secondary alcohol 24 (5.3 mg, quant.) as a colorless oil. 24: R_f = 0.65 (1:2 benzene/acetone); [α]_D²⁵ +12 (c 1.7, CHCl₃); IR (CHCl₃): 3515, 3026, 2985, 2939, 2907, 2875, 2859, 1732, 1656, 1465, 1375, 1250, 1096, 1045, 846, 668 cm^–1; ¹H NMR (400 MHz, CDCl₃): δ 8.28 [8.06]¹ (s, 1H), 7.68–7.63 (m, 4H), 7.47–7.36 (m, 6H), 7.28 (dd, J = 15.2, 9.8 Hz, 1H), 6.49 [7.16]¹ (d, J = 14.0 Hz, 1H), 6.27–6.12 (m, 2H), 5.80 (d, J = 15.2 Hz, 1H), 5.02–4.95 (m, 2H), 4.79 (dd, J = 10.0, 2.6 Hz, 1H), 4.32 [4.59]² (dd, J = 11.0, 4.2 Hz, 1H), 4.01–3.88 (m, 2H), 4.76 (dd, J = 10.4, 4.1 Hz, 1H), 3.62 (dd, J = 10.4, 7.8 Hz, 1H), 3.42 (t, J = 4.6 Hz, 1H), 3.23 (q, J = 7.1 Hz, 1H), 3.01 [3.05]¹ (s, 3H), 2.58 (m, 1H), 2.42 (m, 1H), 2.38 [2.35]² (s, 6H), 2.36 (m, 1H), 2.08 [2.07]¹ [2.06]² (s, 3H), 1.99 (m, 1H), 1.89–1.67 (m, 4H), 1.65–1.49 (m, 3H), 1.30 (d, J = 7.1 Hz, 3H), 1.25 (s, 1H), 1.05 (s, 9H), 1.02 (d, J = 6.8 Hz, 3H), 1.00 (d, J = 6.9 Hz, 3H), 0.97 (d, J = 6.6 Hz, 3H), 0.95 (d, J = 6.5 Hz, 3H), 0.90 [0.88]² (s, 9H), 0.81 (d, J = 6.9 Hz, 3H), 0.05 (s, 6H); ¹³C NMR (100 MHz, CDCl₃): δ 172.4, 170.5, 167.3, 162.1 [161.9]¹, 144.8, 141.0, 135.6 (4C), 132.7 (2C), 130.4, 130.0 (2C), 129.4, 127.7 (4C), 119.7, 110.5 [112.1]¹, 78.8, 76.5, 75.6, 72.1, 68.8, 66.7, 62.8, 41.5 (2C), 39.5 [39.6]¹, 38.6, 37.1, 37.0, 33.0, 30.9, 30.1, 29.3, 27.6, 26.9 (3C), 26.1 (3C), 20.9, 19.4, 19.1, 18.4, 16.6, 15.7, 15.4, 13.3, 9.6, −3.8, −4.1; chemical shifts of the minor diastereomers are within parentheses as follows: []¹, 1.9:1 at C34 stereoisomers; []², S/R = 5.0:1 at C29 DMAla moiety; HRMS (ESI) m/z: 1021.6356 (calcd for C₅₇H₉₃N₂O₁₀Si₂ [M + H]⁺, Δ −1.3 mmu).

TMSer Ester 24a

A solution of secondary alcohol 24 (5.3 mg, 5.2 μmol), N,N,O-trimethyl-l-serine (7.7 mg, 52 μmol), and DMAP (7.8 mg, 64 μmol) in dry CH₂Cl₂ (0.17 mL) was added EDC·HCl (11 mg, 57 μmol). After stirring at room temperature for 2.5 h, the reaction mixture was quenched with sat. NaHCO₃ aq (1 mL) at 0 °C and extracted with EtOAc (1 mL × 4). The combined extracts were washed with brine, dried with Na₂SO₄, and concentrated. The crude material was purified with SiO₂ column chromatography (FL60D 0.5 g, CHCl₃/acetone = 1/0, 10/1 to 0/1) to give TMSer ester 24a (4.1 mg, 69%) as a colorless oil. 24a: R_f = 0.12 (3:1 CHCl₃/acetone); ¹H NMR (400 MHz, CDCl₃): δ 8.30 [8.08]¹ (s, 1H), 7.67–7.64 (m, 4H), 7.46–7.37 (m, 6H), 7.22 (dd, J = 15.4, 10.8 Hz, 1H), 6.49 [7.16]¹ (d, J = 14.1 Hz, 1H), 6.18 (dd, J = 14.2, 10.8 Hz, 1H), 6.03 (m, 1H), 5.80 (d, J = 15.4 Hz, 1H), 5.13 (m, 1H), 5.02–4.96 (m, 2H), 4.80 (dd, J = 10.0, 2.6 Hz, 1H), 4.33 (dd, J = 11.0, 4.2 Hz, 1H), 3.98 (br d, J = 11.0 Hz, 1H), 3.67 (dd, J = 10.2, 5.5 Hz, 1H), 3.36–3.50 (m, 3H), 3.43 (t, J = 4.5 Hz, 1H), 3.32 (dd, J = 8.9, 6.5 Hz, 1H), 3.29 (s, 3H), 3.23 (q, J = 7.1 Hz, 1H), 3.02 [3.06]¹ (s, 3H), 2.65–2.52 (m, 2H), 2.42 (m, 1H), 2.38 [2.36]² (s, 6H), 2.29 [2.33]³ (s, 6H), 2.09 [2.08]¹ (s, 3H), 2.07–2.01 (m, 2H), 1.89–1.80 (m, 2H), 1.68–1.50 (m, 4H), 1.31 [1.28]¹ (d, J = 7.1 Hz, 3H), 1.07 (s, 9H), 1.04 (d, J = 6.8 Hz, 3H), 1.02 (d, J = 7.0 Hz, 3H), 0.98 (d, J = 7.0 Hz, 3H), 0.96 (d, J = 7.1 Hz, 3H), 0.94 (d, J = 6.9 Hz, 3H), 0.90 [0.91]² (s, 9H), 0.04 [0.03]² (s, 6H); chemical shifts of the minor diastereomers are within parentheses as follows: []¹, 1.5:1 at C34 stereoisomers; []², S/R = 5.0:1 at C29 DMAla moiety; []³, S/R = 5.8:1 at C7 TMSer moiety; ¹³C NMR (CDCl₃, 100 MHz): δ 172.5, 170.5, 170.2, 162.1, 160.8, 144.3, 139.0, 135.6 (4C), 133.5, 130.9, 129.7 (2C), 129.3, 127.7 (4C), 125.4, 112.1, 110.5, 78.8, 74.5, 72.1, 71.1, 67.2, 66.8, 65.2, 62.9, 60.4, 59.0, 42.0 (2C), 41.6 (2C), 38.7, 37.1, 37.0, 34.7, 33.0, 30.1, 30.0, 27.6, 26.9 (3C), 26.1 (3C), 20.9, 19.2, 18.4, 16.6, 15.7, 15.4, 14.2, 13.1, 11.4, 9.7, −3.8, −4.1; HRMS (ESI) m/z: 1150.7179 (calcd for C₆₃H₁₀₄N₃O₁₂Si₂ [M + H]⁺, Δ −2.0 mmu).

ApA Analogue 5

TMSer ester 24a (1.2 mg, 1.0 μmol) was dissolved in a 5:3:7 mixture of hydrogen fluoride pyridine, pyridine, and dry THF (0.5 mL) at 0 °C. After being stirred for 18 h at room temperature, the reaction mixture was poured into sat. NaHCO₃ aq (2 mL) at 0 °C and extracted with EtOAc (2 mL × 4). The combined extracts were washed with brine, dried with Na₂SO₄, and concentrated. The crude material was purified with SiO₂ column chromatography (FL60D 0.5 g, CHCl₃/acetone = 1/0, 10/1 to 0/1) to give ApA analogue 5 (0.4 mg, 50%) as a colorless oil. For bioassay, a part of the sample was further purified with reversed-phase HPLC [Develosil ODS-HG-5 (ϕ 20 × 250 mm), MeOH/20 mM aq NH₄OAc (74/26), 5 mL/min, UV254 nm, t_R 31.3–35.3 min]. As with previous synthetic studies of aplyronines,(4) epimerization of the TMSer ester moiety was observed during condensation. While compound 5 was separable as a mixture of two stereoisomers by reversed-phase HPLC, bioassays were carried out with a mixture of four stereoisomers. 5: R_f = 0.05 (1:1 CHCl₃/acetone); ¹H NMR (400 MHz, CDCl₃): δ 8.29 [8.08]¹ (s, 1H), 7.21 (m, 1H), 6.48 [7.16]¹ (d, J = 14.0 Hz, 1H), 6.32–6.17 (m, 2H), 5.82 (d, J = 15.3 Hz, 1H), 5.43 (m, 1H), 5.02–4.94 (m, 2H), 4.78 (dd, J = 10.0, 1.9 Hz, 1H), 4.32–4.24 (m, 2H), 4.18 (dd, J = 11.0, 5.0 Hz, 1H), 4.23–4.10 (m, 2H), 3.94 (dd, J = 10.2, 6.2 Hz, 1H), 3.73 (m, 1H), 3.65 (s, 1H), 3.54–3.40 (m, 2H), 3.36 [3.07]³ (s, 3H), 3.19 (s, 1H), 3.03 [3.07]¹ (s, 3H), 2.62 [2.63]² (s, 6H), 2.67–2.45 (m, 2H), 2.36 [2.35]³ (s, 6H), 2.35 (m, 1H), 2.07 [2.05]¹ (s, 3H), 2.08–1.95 (m, 2H), 1.90–1.77 (m, 2H), 1.70–1.41 (m, 4H), 1.31 (d, J = 6.9 Hz, 3H), 1.01 (d, J = 6.9 Hz, 3H), 1.00 (d, J = 7.0 Hz, 3H), 0.97 (d, J = 6.8 Hz, 3H), 0.96 (d, J = 6.8 Hz, 3H), 0.95 (d, J = 6.9 Hz, 3H); chemical shifts of the minor diastereomers are within parentheses as follows: []¹, 1.5:1 at C34 stereoisomers; []², S/R = 2.0:1 at C29 DMAla moiety; []³, S/R = 2.0:1 at C7 TMSer moiety; HRMS (ESI) m/z: 820.4929 (calcd for C₄₁H₇₁N₃NaO₁₂ [M + Na]⁺, Δ −0.7 mmu).

ApC Analogue 6

Prepared from secondary alcohol 24 as in the case with ApA analogue 5 in 90% yield. In addition, this can be prepared from ester 21a using the same method in quantitative yield. For bioassay, a part of the sample was further purified with reversed-phase HPLC [Develosil ODS-HG-5 (ϕ 20 × 250 mm), MeOH/20 mM aq NH₄OAc (70/30), 5 mL/min, UV254 nm, t_R 30.0–33.8 min]. The major side-product of the esterification of 6 was the mono-C9-TMSer ester (ca. 40% yield). Analogue 6 was obtained as a mixture of stereoisomers at the C7 TMSer and C29 DMAla ester moieties and was not separable by reversed-phase HPLC. Bioassays were carried out with mixture of stereoisomers. 6: R_f = 0.55 (3:1 CHCl₃/MeOH); ¹H NMR (400 MHz, CDCl₃): δ 8.28 [8.07]¹ (s, 1H), 7.25 (m, 1H), 6.47 [7.14]¹ (d, J = 14.0 Hz, 1H), 6.33–6.17 (m, 2H), 5.82 (d, J = 15.4 Hz, 1H), 4.93–5.04 (m, 2H), 4.78 (dd, J = 10.0, 2.4 Hz, 1H), 4.29 (dd, J = 11.0, 4.6 Hz, 1H), 4.20 (m, 1H), 3.78 (m, 1H), 3.71–3.62 (m, 2H), 3.66 (s, 1H), 3.49 (s, 1H), 3.45 (m, 1H), 3.20 (m, 1H), 3.19 (s, 1H), 3.03 [3.06]¹ (s, 3H), 2.60–2.47 (m, 2H), 2.36 [2.35]² (s, 6H), 2.35 (m, 1H), 2.07 (s, 3H), 2.05–1.98 (m, 3H), 1.85 (m, 1H), 1.79–1.50 (m, 4H), 1.30 (d, J = 6.9 Hz, 3H), 1.01 (d, J = 6.9 Hz, 3H), 0.99 (d, J = 7.1 Hz, 3H), 0.98 (d, J = 7.0 Hz, 3H), 0.90 (d, J = 6.8 Hz, 3H), 0.86 (d, J = 6.9 Hz, 3H); chemical shifts of the minor diastereomers are within parentheses as follows: []¹, 1.9:1 at C34 stereoisomers; []², S/R = 2.1:1 at C29 DMAla moiety; ¹³C NMR (100 MHz, CDCl₃): δ 170.7, 170.5, 162.2, 161.0, 145.4, 144.9, 140.4, 131.0, 129.3, 110.7, 73.9, 72.1, 70.5, 67.7, 66.6, 62.9, 41.6 (2C), 39.7, 39.0, 37.0, 35.7, 35.2, 34.8, 33.1, 29.7, 27.7, 25.9, 20.9, 19.4, 16.8, 15.4, 14.7, 13.7, 9.7; HRMS (ESI) m/z: 691.4124 (calcd for C₃₅H₆₀N₂NaO₁₀ [M + Na]⁺, Δ −2.1 mmu).

Ester 21a

Prepared from carboxylic acid 21 and the C23–C34 segment 16 as in the case with ester 20a in 68% yield. 21a: R_f = 0.68 (2:1 CHCl₃/acetone); [α]_D²⁵ −16 (c 0.50, CHCl₃); IR (CHCl₃): 3028, 2957, 2930, 2857, 1730, 1695, 1655, 1471, 1461, 1251, 1108, 1078, 836, 703 cm^–1; ¹H NMR (400 MHz, CDCl₃): δ 8.28 [8.06]¹ (s, 1H), 7.65–7.63 (m, 4H), 7.42–7.31 (m, 6H), 7.23 (dd, J = 15.4, 9.8 Hz, 1H), 6.49 [7.16]¹ (d, J = 14.0 Hz, 1H), 6.16 (dd, J = 15.2, 9.8 Hz, 1H), 6.11 (m, 1H), 5.77 (d, J = 15.4 Hz, 1H), 4.98 (m, 1H), 4.97 (dd, J = 14.0, 8.9 Hz, 1H), 4.79 (d, J = 9.8 Hz, 1H), 4.33 (dd, J = 11.0, 4.3 Hz, 1H), 3.98–3.84 (m, 2H), 3.62 (m, 1H), 3.48 (dd, J = 10.2, 6.4 Hz, 1H), 3.43 (t, J = 4.7 Hz, 1H), 3.22 (q, J = 7.1 Hz, 1H), 3.01 [3.05]¹ (s, 3H), 2.56 (m, 1H), 2.37 [2.35]² (s, 6H), 2.32–2.20 (m, 2H), 2.07 [2.06]¹ (s, 3H), 2.01–1.84 (m, 3H), 1.66–1.41 (m, 5H), 1.30 (d, J = 7.1 Hz, 3H), 1.05 (s, 9H), 1.02 (d, J = 6.8 Hz, 3H), 0.96 (d, J = 7.0 Hz, 3H), 0.94 (d, J = 7.0 Hz, 3H), 0.89 (d, J = 6.6 Hz, 3H), 0.88 (s, 6H), 0.85 (d, J = 7.4 Hz, 3H), 0.83 (s, 12H), 0.04 (s, 3H), 0.03 (s, 3H), −0.01 (s, 3H), −0.03 (s, 3H); ¹³C NMR (100 MHz, CDCl₃): δ 172.5, 170.5, 167.4, 162.1 [160.9]¹, 145.1, 141.8, 135.6 (4C), 133.8 (2C), 130.2, 129.6 (2C), 129.3, 127.6 (4C), 119.3, 110.5 [112.1]¹, 78.8, 76.5, 72.7, 72.1, 65.9, 62.3, 60.4, 41.5 [41.4]² (2C), 41.3, 37.0, 36.6, 36.2, 33.0, 31.6, 30.1, 28.6, 27.6, 26.9 (3C), 26.1 (3C), 25.8 (3C), 21.0, 19.3, 18.4, 18.0, 16.7, 15.7, 15.4, 14.2, 11.9, 9.6, −3.8, −4.1, −4.3, −4.6; chemical shifts of the minor diastereomers are within parentheses as follows: []¹, 1.9:1 at C34 stereoisomers; []², S/R = 7.2:1 at C29 DMAla moiety; HRMS (ESI) m/z: 1135.7201 (calcd for C₆₃H₁₀₇N₂O₁₀Si₃ [M + H]⁺, Δ −2.7 mmu).

Bis-TMSer Ester 7

To a solution of ApC analogue 6 (2.2 mg, 3.1 μmol), N,N,O-trimethyl-l-serine (5.3 mg, 36 μmol), and DMAP (4.4 mg, 36 μmol) in dry CH₂Cl₂ (0.15 mL) was added EDC·HCl (6.3 mg, 33 μmol). After stirring at room temperature for 22 h, the reaction mixture was quenched with sat. NaHCO₃ aq (1 mL) at 0 °C and extracted with EtOAc (1 mL × 3). The combined extracts were washed with brine, dried with Na₂SO₄, and concentrated. The crude material was purified with a reversed-phase HPLC [Develosil ODS-HG-5 (ϕ 20 × 250 mm), MeOH/20 mM aq NH₄OAc (75/25), 5 mL/min, UV254 nm, t_R 23.8–27.4 min] to give bis-TMSer ester 7 (0.6 mg, 21%, S/R = 1.6:1 at C34 enamide moiety) as a colorless oil. The eluate was freeze-dried, dissolved in water, and freeze-dried again twice to remove remaining ammonium acetate. Analogue 7 was obtained as a mixture of stereoisomers at the C7 TMSer and C29 DMAla ester moieties and was not separable by reversed-phase HPLC. Bioassays were carried out with mixture of stereoisomers. 7: R_f = 0.73 (5:1 CHCl₃/MeOH); ¹H NMR (400 MHz, CDCl₃): δ 8.29 [8.08] (s, 1H), 7.24 (m, 1H), 6.48 [7.15] (d, J = 14.0 Hz, 1H), 6.24 (dd, J = 15.2, 11.0 Hz, 1H), 6.09 (m, 1H), 5.82 (d, J = 15.4 Hz, 1H), 5.07–4.95 (m, 3H), 4.78 (dd, J = 9.8, 1.9 Hz, 1H), 4.30 (m, 1H), 4.20 (m, 1H), 4.02 (m, 2H), 3.64 (dd, J = 8.9, 2.8 Hz, 2H), 3.61 (dd, J = 12.3, 6.2 Hz, 2H), 3.56 (s, 6H), 3.44–3.32 (m, 2H), 3.24–3.19 (m, 2H), 3.03 [3.07]^a (s, 3H), 2.65–2.45 (m, 3H), 2.40–2.32 (m, 18H), 2.16–1.92 (m, 3H), 2.07 (s, 3H), 1.92–1.35 (m, 6H), 1.30 (d, J = 7.1 Hz, 3H), 1.02 (d, J = 6.7 Hz, 3H), 0.97 (d, J = 7.4 Hz, 3H), 0.95 (d, J = 7.4 Hz, 3H), 0.86 (d, J = 6.7 Hz, 3H), 0.82 (d, J = 6.6 Hz, 3H); chemical shifts of the minor diastereomers are within parentheses (square blankets); HRMS (ESI) m/z: 949.5694 (calcd for C₄₇H₈₂N₄NaO₁₄ [M + Na]⁺, Δ −2.5 mmu).

Ester 23a

Prepared from carboxylic acid 23 and the C23–C34 segment 16 as in the case with ester 20a in 65% yield. 23a: R_f = 0.61 (2:1 CHCl₃/acetone); [α]_D²² −20 (c 0.34, CHCl₃); IR (CHCl₃): 3028, 2955, 2929, 2857, 1726, 1696, 1655, 1471, 1301, 1097, 836 cm^–1; ¹H NMR (400 MHz, CDCl₃): δ 8.28 [8.06]¹ (s, 1H), 7.21 (dd, J = 15.4, 11.0 Hz, 1H), 6.48 [7.16]¹ (d, J = 14.0 Hz, 1H), 6.26 (dd, J = 15.4, 11.0 Hz, 1H), 6.06 (dd, J = 15.4, 8.2 Hz, 1H), 5.80 (d, J = 15.4 Hz, 1H), 5.00 (m, 1H), 4.97 (dd, J = 14.0, 9.3 Hz, 1H), 4.79 (dd, J = 9.8, 3.0 Hz, 1H), 4.42 (dd, J = 11.0, 4.4 Hz, 1H), 3.92 (m, 1H), 3.68 (dd, J = 9.8, 6.0 Hz, 2H), 3.64 (dd, J = 9.8, 6.0 Hz, 2H), 3.42 (br t, J = 4.5 Hz, 1H), 3.22 (q, J = 7.2 Hz, 1H), 3.01 [3.04]^a (s, 3H), 2.55 (m, 1H), 2.45 (m, 1H), 2.37 [2.35]² (s, 6H), 2.04 (s, 3H), 2.00 (m, 1H), 1.87 (m, 1H), 1.65–1.33 (m, 5H), 1.30 (d, J = 7.1 Hz, 3H), 1.02 (d, J = 6.8 Hz, 3H), 0.95 (d, J = 6.8 Hz, 3H), 0.94 (d, J = 6.8 Hz, 3H), 0.88 (d, J = 6.8 Hz, 3H), 0.88 (s, 27H), 0.03 (s, 18H); ¹³C NMR (100 MHz, CDCl₃): δ 172.5, 170.5, 167.1, 162.1 [160.8]¹, 145.0, 142.8, 129.9, 129.4, 119.9, 110.5 [112.1]¹, 78.8, 76.5, 72.2, 62.9, 62.5 (2C), 60.4, 48.0, 41.5 [41.4]² (2C), 37.1, 37.0, 36.2, 33.0, 30.3, 28.6, 27.6, 26.1 (6C), 25.9 (3C), 20.9, 19.4, 18.3, 18.2 (2C), 16.7, 15.4, 14.2, 9.7, −3.8, −4.2, −5.4 (2C), −5.5 (2C); chemical shifts of the minor diastereomers are within parentheses as follows: []¹, 1.9:1 at C34 stereoisomers; []², S/R = 9.0:1 at C29 DMAla moiety; HRMS (ESI) m/z: 1005.6428 (calcd for C₅₁H₉₈NaN₂O₁₀Si₃ [M + Na]⁺, Δ +0.6 mmu).

Triol 25

Prepared from ester 23a as in the case with ApA analogue 5 in 80% yield. 25: R_f = 0.27 (10:1 CHCl₃/MeOH); ¹H NMR (400 MHz, CDCl₃): δ 8.28 [8.07]¹ (s, 1H), 7.24 (dd, J = 15.4, 10.8 Hz, 1H), 6.48 [7.15]¹ (d, J = 14.1 Hz, 1H), 6.32 (dd, J = 15.4, 10.8 Hz, 1H), 6.10 (dd, J = 15.4, 8.1 Hz, 1H), 5.85 (d, J = 15.4 Hz, 1H), 5.00 (m, 1H), 4.98 (dd, J = 14.1, 9.4 Hz, 1H), 4.78 (dd, J = 9.5, 2.4 Hz, 1H), 4.29 (dd, J = 11.2, 5.5 Hz, 1H), 4.20 (dd, J = 11.2, 4.7 Hz, 1H), 3.83 (dd, J = 10.8, 5.4 Hz, 2H), 3.80 (dd, J = 11.2, 6.1 Hz, 2H), 3.18 (q, J = 7.6 Hz, 1H), 3.15 (m, 1H), 3.02 [3.07]¹ (s, 3H), 2.63 (m, 1H), 2.54 (m, 1H), 2.37 [2.35]² (s, 6H), 2.07 (s, 3H), 2.06–1.35 (m, 10H), 1.30 (d, J = 7.1 Hz, 3H), 1.02 (d, J = 6.8 Hz, 3H), 1.01 (d, J = 6.8 Hz, 3H), 0.97 (d, J = 7.0 Hz, 3H), 0.95 (d, J = 6.9 Hz, 3H); ¹³C NMR (100 MHz, CDCl₃): δ 172.6, 170.6, 167.2, 162.2 [161.0]¹, 144.7, 141.2, 130.6, 129.4, 120.6, 110.7 [112.3]¹, 78.1, 72.2, 70.5, 66.6, 64.5 (2C), 63.0, 46.9, 41.6 [41.5]² (2C), 36.9, 35.7, 35.2, 33.1, 29.7, 27.6, 25.9, 20.9, 19.4, 16.8, 15.4, 14.7, 9.7; chemical shifts of the minor diastereomers are within parentheses as follows: []¹, 1.7:1 at C34 stereoisomers; []², S/R = 6.8:1 at C29 DMAla moiety; HRMS (ESI) m/z: 641.3981 (calcd for C₃₃H₅₇N₂O₁₀ [M + H]⁺, Δ −2.7 mmu).

Bis-TMSer Ester 8

Prepared from ester 25 as in the case with bis-TMSer ester 7 in 20% yield (S/R = 1.7:1 at C34 enamide moiety), except for the HPLC solvent system [MeOH/20 mM aq NH₄OAc (67/33), t_R 71.9–78.1 min]. Analogue 8 was obtained as a mixture of stereoisomers at the C7 and C7′ TMSer and C29 DMAla ester moieties and was not separable by reversed-phase HPLC. Bioassays were carried out with mixture of stereoisomers. 8: R_f = 0.77 (5:1 CHCl₃/MeOH); ¹H NMR (600 MHz, CDCl₃): δ 8.29 [8.08] (s, 1H), 7.23 (dd, J = 15.4, 10.9 Hz, 1H), 6.48 [7.15] (d, J = 14.1 Hz, 1H), 6.32 (dd, J = 15.5, 10.9 Hz, 1H), 6.01 (dd, J = 15.5, 7.9 Hz, 1H), 5.87 (d, J = 15.4 Hz, 1H), 4.99 (m, 1H), 4.96 (dd, J = 14.1, 9.4 Hz, 1H), 4.78 (dd, J = 9.9, 2.8 Hz, 1H), 4.32 (dd, J = 11.0, 5.6 Hz, 1H), 4.28–4.18 (m, 5H), 3.63 (dd, J = 10.5, 8.4 Hz, 2H), 3.60 (dd, J = 9.6, 6.5 Hz, 2H), 3.41 (t, J = 6.6 Hz, 2H), 3.35 (s, 6H), 3.22 (q, J = 7.2 Hz, 1H), 3.19 (dd, J = 7.2, 4.8 Hz, 1H), 3.03 [3.07] (s, 3H), 2.96 (m, 1H), 2.55 (m, 1H), 2.37 (s, 18H), 2.27 (s, 3H), 1.98 (m, 1H), 1.84 (m, 1H), 1.75–1.40 (m, 6H), 1.31 (d, J = 7.2 Hz, 3H), 1.02 (d, J = 6.8 Hz, 3H), 1.01 (d, J = 6.8 Hz, 3H), 0.97 (d, J = 6.9 Hz, 3H), 0.95 (d, J = 6.9 Hz, 3H); ¹³C NMR (150 MHz, CDCl₃): δ 172.6, 170.4 (3C), 167.7, 162.2, 144.2, 138.9, 131.3, 129.4, 121.2, 110.5, 77.7, 72.2, 70.9 (2C), 67.0 (2C), 66.6, 63.3, 62.9, 59.2 (2C), 42.2 (6C), 41.6 (2C), 41.5, 37.1, 37.0, 35.7, 35.1, 33.1, 29.8, 27.6, 21.0, 19.4, 16.8, 15.5, 14.7, 9.8; chemical shifts of the minor diastereomers are within parentheses (square blankets); HRMS (ESI) m/z: 921.5400 (calcd for C₄₅H₇₈N₄NaO₁₄ [M + Na]⁺, Δ −0.7 mmu).

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Corresponding Authors
- Hideo Kigoshi - Graduate School of Pure and Applied Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba 305-8571, Japan; http://orcid.org/0000-0003-1554-5129; Email: [email protected]
- Masaki Kita - Graduate School of Pure and Applied Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba 305-8571, Japan; Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa, Nagoya 464-8601, Japan; http://orcid.org/0000-0001-6685-4461; Email: [email protected]
Authors
- Kentaro Futaki - Graduate School of Pure and Applied Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba 305-8571, Japan
- Momoko Takahashi - Graduate School of Pure and Applied Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba 305-8571, Japan
- Kenta Tanabe - Graduate School of Pure and Applied Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba 305-8571, Japan
- Akari Fujieda - Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa, Nagoya 464-8601, Japan
Notes
The authors declare no competing financial interest.

Acknowledgments

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This work is supported in part by JSPS grants (18H04613 and 19H02839 to M.K. and 26242073 to H.K.) and JSPS A3 Foresight Program. Supports were also provided by PRESTO, JST (JPMJPR1535), and the Naito Foundation. We also thank the Kaneka Corporation for their gift of methyl (S)-3-hydroxy-2-methylpropionate.

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Hirayama, Y.; Yamagishi, K.; Suzuki, T.; Kawagishi, H.; Kita, M.; Kigoshi, H. Analysis of the aplyronine A-induced protein–protein interaction between actin and tubulin by surface plasmon resonance. Bioorg. Med. Chem. 2016, 24, 2809– 2814, DOI: 10.1016/j.bmc.2016.04.049
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(a) Yoneda, K.; Hu, Y.; Kita, M.; Kigoshi, H. 6-Amidopyrene as a label-assisted laser desorption/ionization (LALDI) enhancing tag: development of photoaffinity pyrene derivative. Sci. Rep. 2015, 5, 17853, DOI: 10.1038/srep17853
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9a
6-Amidopyrene as a label-assisted laser desorption/ionization (LA-LDI) enhancing tag: development of photoaffinity pyrene derivative
Yoneda, Kozo; Hu, Yaping; Kita, Masaki; Kigoshi, Hideo
Scientific Reports (2015), 5 (), 17853CODEN: SRCEC3; ISSN:2045-2322. (Nature Publishing Group)
Pyrene-conjugated compds. are detected by label-assisted laser desorption/ionization mass spectrometry (LA-LDI MS) without matrixes. We found that 6-amidopyrene derivs. were highly detectable by the LDI MS instrument equipped with a 355 nm laser. In a certain case of a 6-amidopyrene deriv., a mol. ion peak [M]+· and a characteristic fragment ion peak [M-42]+· were detected in an amt. of only 10 fmol. The latter peak, corresponding to the 6-aminopyrene fragment, might be generated in situ by the removal of ketene (CH2=C=O) from the parent mol. A photoaffinity amidopyrene deriv. of an antitumor macrolide aplyronine A (ApA-PaP) was synthesized, which showed potent cytotoxicity and actin-depolymg. activity. In an LDI MS anal. of the MeOH- and water-adducts of ApA-PaP, oxime N-O bonds as well as amidopyrene N-acetyl moieties were preferentially cleaved, and their internal structures were confirmed by MS/MS anal. Amidopyrene moiety might enhance fragmentation and stabilize the cleaved fragments by intramol. or intermol. weak interactions including hydrogen bonding. Our chem. probe methods might contribute to a detailed anal. of binding modes between various ligands and target biomacromols. that include multiple and weak interactions.
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(b) Kita, M.; Hirayama, Y.; Yamagishi, K.; Yoneda, K.; Fujisawa, R.; Kigoshi, H. Interactions of the antitumor macrolide aplyronine A with actin and actin-related proteins established by its versatile photoaffinity derivatives. J. Am. Chem. Soc. 2012, 134, 20314– 20317, DOI: 10.1021/ja310495p
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(c) Kita, M.; Hirayama, Y.; Sugiyama, M.; Kigoshi, H. Development of highly cytotoxic and actin-depolymerizing biotin derivatives of aplyronine A, an antitumor macrolide of marine origin. Angew. Chem., Int. Ed. 2011, 50, 9871– 9874, DOI: 10.1002/anie.201103802
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(a) Kita, M.; Yamagishi, K.; Tsuchiya, K.; Seguchi, Y.; Nakane, H.; Kigoshi, H. Development of photoaffinity derivatives of the antitumor macrolide aplyronine A, a PPI-inducer between actin and tubulin. Bioorg. Med. Chem. 2017, 25, 6322– 6331, DOI: 10.1016/j.bmc.2017.09.044
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Structural basis of swinholide A binding to actin
Klenchin, Vadim A.; King, Ryan; Tanaka, Junichi; Marriott, Gerard; Rayment, Ivan
Chemistry & Biology (2005), 12 (3), 287-291CODEN: CBOLE2; ISSN:1074-5521. (Cell Press)
Marine toxins targeting the actin cytoskeleton represent a new and promising class of anti-cancer compds. Here we present a 2.0 Å resoln. structure of swinholide A, a marine macrolide, bound to two actin mols. The structure demonstrates that the actin dimer in the complex does not represent a physiol. relevant entity, for the two actin mols. do not interact with each other. The swinholide A actin binding site is the same as that targeted by toxins of the trisoxazole family and numerous actin binding proteins, highlighting the importance of this site in actin polymn. The obsd. structure reveals the mechanism of action of swinholide A and provides a structural framework about which to design new agents directed at the cytoskeleton.
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(a) Suenaga, K.; Kimura, T.; Kuroda, T.; Matsui, K.; Miya, S.; Kuribayashi, S.; Sakakura, A.; Kigoshi, H. Synthesis and biological activity of mycalolide analogs. Tetrahedron 2006, 62, 8278– 8290, DOI: 10.1016/j.tet.2006.06.046
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Study of the Interaction between Actin and Antitumor Substance Aplyronine A with a Novel Fluorescent Photoaffinity Probe
Kuroda, Takeshi; Suenaga, Kiyotake; Sakakura, Akira; Handa, Tomohisa; Okamoto, Kazuhito; Kigoshi, Hideo
Bioconjugate Chemistry (2006), 17 (2), 524-529CODEN: BCCHES; ISSN:1043-1802. (American Chemical Society)
The interaction between actin and aplyronine A, a potent antitumor and actin-depolymg. substance of marine origin, was investigated by photoaffinity labeling expts. Photoaffinity probes consisting of a side-chain portion of aplyronine A as a ligand, a diazirine moiety as a photoaffinity group, and a fluorophore as a detecting group were synthesized. Photolabeling expts. between actin and the probe were carried out. Actin was successfully photolabeled by the fluorescent probe and visualized clearly. The present results provide the first chem. evidence for the direct interaction between actin and the side-chain portion of aplyronine A.
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(a) Kobayashi, K.; Fujii, Y.; Hirayama, Y.; Kobayashi, S.; Hayakawa, I.; Kigoshi, H. Design, synthesis, and biological evaluations of aplyronine A–mycalolide B hybrid compound. Org. Lett. 2012, 14, 1290– 1293, DOI: 10.1021/ol300182r
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Zampella, A.; D’Auria, M. V. Stereoselective synthesis of (2R,3R,4R)-3-hydroxy-2,4,6-trimethylheptanoic acid and determination of the absolute stereochemistry of the natural product from callipeltin A. Tetrahedron: Asymmetry 2002, 13, 1237– 1239, DOI: 10.1016/s0957-4166(02)00328-2
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(a) Blakemore, P. R.; Cole, W. J.; Kocienski, P. J.; Morley, A. A stereoselective synthesis of trans-1,2-disubstituted alkenes based on the condensation of aldehydes with metallated 1-phenyl-1H-tetrazol-5-yl sulfones. Synlett 1998, 26– 28, DOI: 10.1055/s-1998-1570
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A stereoselective synthesis of trans-1,2-disubstituted alkenes based on the condensation of aldehydes with metalated 1-phenyl-1H-tetrazol-5-yl sulfones
Blakemore, Paul R.; Cole, William J.; Kocienski, Philip J.; Morley, Andrew
Synlett (1998), (1), 26-28CODEN: SYNLES; ISSN:0936-5214. (Georg Thieme Verlag)
The reaction of metalated 1-phenyl-1H-tetrazol-5-yl sulfones and aldehydes gives good yields and stereoselectivity of trans-1,2-disubstituted alkenes with KN(SiMe3)2 or NaN(SiMe3)2 as base and DME as solvent.
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The modified Julia olefination: alkene synthesis via the condensation of metallated heteroarylalkylsulfones with carbonyl compounds
Blakemore, Paul R.
Journal of the Chemical Society, Perkin Transactions 1 (2002), (23), 2563-2585CODEN: JCSPCE; ISSN:1472-7781. (Royal Society of Chemistry)
A review. The classical and modified Julia olefination in the title synthesis were reviewed and discussed in details.
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Kita, M.; Oka, H.; Usui, A.; Ishitsuka, T.; Mogi, Y.; Watanabe, H.; Tsunoda, M.; Kigoshi, H. Total synthesis of mycalolides A and B through olefin metathesis. Angew. Chem., Int. Ed. 2015, 54, 14174– 14178, DOI: 10.1002/anie.201507795
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Total Synthesis of Mycalolides A and B through Olefin Metathesis
Kita, Masaki; Oka, Hirotaka; Usui, Akihiro; Ishitsuka, Tomoya; Mogi, Yuzo; Watanabe, Hidekazu; Tsunoda, Masaki; Kigoshi, Hideo
Angewandte Chemie, International Edition (2015), 54 (47), 14174-14178CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
An asym. total synthesis of the trisoxazole marine macrolides mycalolides A and B is described. This synthesis involves the convergent assembly of highly functionalized C1-C19 trisoxazole and C20-C35 side-chain segments through the use of olefin metathesis and esterification as well as Julia-Kocienski olefination and enamide formation as key steps.
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(a) Dess, D. B.; Martin, J. C. Readily accessible 12-I-5 oxidant for the conversion of primary and secondary alcohols to aldehydes and ketones. J. Org. Chem. 1983, 48, 4155– 4156, DOI: 10.1021/jo00170a070
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Readily accessible 12-I-5 oxidant for the conversion of primary and secondary alcohols to aldehydes and ketones
Dess, D. B.; Martin, J. C.
Journal of Organic Chemistry (1983), 48 (22), 4155-6CODEN: JOCEAH; ISSN:0022-3263.
Oxidn. of 2-IC6H4CO2H with KBrO3 in aq. H2SO4, followed by treatment of the oxidn. product with Ac2O gave 87% periodinane I, a 10-I-5 species (i.e., 10 valence electrons are formally involved in binding 5 ligands to the central iodine atom). I reacted rapidly with primary or secondary alcs. at room temp. to give the corresponding aldehydes or ketones in high yield. Excess I does not further oxidize the aldehyde or ketone under the reaction conditions. The reaction is strongly catalyzed by excess alc. or strong acid, but is unaffected by pyridine. I oxidizes benzylic alcs. selectively in the presence of satd. alcs. Procedures for sepg. the carbonyl product from the reaction mixt. are mild and simple. Cryst. I is stable at room temp. in the absence of moisture.
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A useful 12-I-5 triacetoxyperiodinane (the Dess-Martin periodinane) for the selective oxidation of primary or secondary alcohols and a variety of related 12-I-5 species
Dess, Daniel B.; Martin, J. C.
Journal of the American Chemical Society (1991), 113 (19), 7277-87CODEN: JACSAT; ISSN:0002-7863.
The stable 10-I-4 species 1-hydroxy-1,3-dihydro-3,3-bis(trifluoromethyl)-1,2-benziodoxole 1-oxide (I) is the ring-closed form of o-iodoxyhexafluorocumyl alc. It is prepd. by the oxidn. of chloroiodinane II with KBrO3 in aq. H2SO4. The x-ray crystal structure of the tetrabutylammonium salt of I showed the unusual feature of an apical, neg. charged oxide ligand. 1,1,1-Triacetoxy-1,1-dihydro-1,2-benziodoxol-3(1H)-one (III) (Dess-Martin Periodinane), derived from the 1-hydroxy-1,2-benziodoxol-3(1H)-one 1-oxide by treatment with Ac2O, is an extremely useful reagent for the conversion of primary and secondary alcs. to aldehydes and ketones at 25 °C. It does not oxidize aldehydes to carboxylic acids under these conditions. It selectively oxidizes alcs. in the presence of furan rings or sulfides and does not react with vinyl ethers. Geraniol is oxidized to geranial without isomerization to nerol. Benzylic or allylic alcs. are selectively oxidized in the presence of satd. alkanols. The alc. oxidn. mechanism is discussed.
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Kita, M.; Yoneda, K.; Hirayama, Y.; Yamagishi, K.; Saito, Y.; Sugiyama, Y.; Miwa, Y.; Ohno, O.; Morita, M.; Suenaga, K.; Kigoshi, H. Fluorescent aplyronine A: Intracellular accumulation and disassembly of actin cytoskeleton in tumor cells. ChemBioChem 2012, 13, 1754– 1758, DOI: 10.1002/cbic.201200385
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LowModeMD - Implicit Low-Mode Velocity Filtering Applied to Conformational Search of Macrocycles and Protein Loops
Labute, Paul
Journal of Chemical Information and Modeling (2010), 50 (5), 792-800CODEN: JCISD8; ISSN:1549-9596. (American Chemical Society)
We present a method for conformational search of complex mol. systems such as macrocycles and protein loops. The method is based on perturbing an existing conformation along a mol. dynamics trajectory using initial at. velocities with kinetic energy concd. on the low-frequency vibrational modes, followed by energy minimization. A novel Chebyshev polynomial filter is used to heavily dampen the high-frequency components of a randomly generated Maxwell-Boltzmann velocity vector. The method is very efficient, even for large systems; it is straightforward to implement and requires only std. force-field energy and gradient evaluations. The results of several computational expts. suggest that the method is capable of efficiently sampling low-strain energy conformations of complex systems with nontrivial nonbonded interaction networks.
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Dario Matulja, Karlo Wittine, Nela Malatesti, Sylvain Laclef, Maris Turks, Maria Kolympadi Markovic, Gabriela Ambrožić, Dean Marković. Marine Natural Products with High Anticancer Activities. Current Medicinal Chemistry 2020, 27 (8) , 1243-1307. https://doi.org/10.2174/0929867327666200113154115
Talia R. Pettigrew, Rachel J. Porter, Stephen J. Walsh, Michael P. Housden, Nelson Y. S. Lam, Jason S. Carroll, Jeremy S. Parker, David R. Spring, Ian Paterson. Total synthesis and biological evaluation of simplified aplyronine analogues as synthetically tractable anticancer agents. Chemical Communications 2020, 56 (10) , 1529-1532. https://doi.org/10.1039/C9CC09050A

Abstract
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Figure 1
Figure 1. Structures of aplyronines and their synthetic analogues.
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Figure 2
Figure 2. Molecular modeling studies of aplyronines. The most stable conformers of ApA [(a), green] and ApB [(b), cyan] on actin are shown. Conformational searches were performed using the Amber12:EHT force-field, in which both actin and the C24–C34 side-chain parts of aplyronines were fixed. In each model, ApA (yellow) in the actin–ApA complex (PDB code: 1WUA) is superimposed.
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Scheme 1
Scheme 1. Synthesis of the C23–C34 Side-chain Part 16^a
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^aReagents and conditions: (a) 5,5′-dithiobis(1-phenyl-1H-tetrazole), tri-n-butylphosphine, THF; (b) m-CPBA, NaHCO₃, CH₂Cl₂; (c) LHMDS, DME, −55 °C to rt; (d) Ca, liq. NH₃, i-PrOH, THF, −78 °C; (e) TBDPSCl, imidazole, DMF; (f) H₂, Pd(OH)₂/C, NaHCO₃, EtOH; (g) N,N-dimethyl-l-alanine, EDC·HCl, DMAP, CH₂Cl₂; (h) aq HCl, DME; (i) NaBH₄, EtOH, 0 °C to rt; (j) TrCl, Et₃N, DMAP, CH₂Cl₂; (k) Ac₂O, pyridine, DMAP; (l) HCOOH, EtOH, 40 °C, then NH₃, aq MeOH; (m) Dess–Martin periodinane, pyridine, CH₂Cl₂, (n) N-methylformamide, PPTS, hydroquinone, MS3A (3 Å molecular sieves), benzene, reflux; (o) NH₄F, MeOH, 60 °C.
Scheme 2
Scheme 2. Synthesis of Conjugated Carboxylic Acids 20, 21, and 23^a
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^aReagents and conditions: (a) Ca, liq. NH₃, i-PrOH, THF, −78 °C; (b) 2,2-dimethoxypropane, CSA, CH₂Cl₂; (c) OsO₄, NMO, acetone–H₂O then NaIO₄; (d) LDA, triethyl 4-phosphonocrotonate, THF, −40 °C; (e) PPTS, MeOH; (f) TBDPSCl, imidazole, DMF; (g) DMSO, Ac₂O, AcOH, 40 °C; (h) LiOH, aq MeOH, w/o THF; (i) TBSCl, imidazole, DMAP, CH₂Cl₂.
Scheme 3
Scheme 3. Synthesis of Aplyronine Analogues 5–8^a
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^aReagents and conditions: (a) 2,4,6-trichlorobenzoyl chloride, Et₃N, THF, then 16, DMAP, toluene; (b) AgNO₃, 2,6-lutidine, THF–H₂O; (c) N,N,O-trimethyl-l-serine, EDC·HCl, DMAP, CH₂Cl₂; (d) HF·pyridine, pyridine, THF (5:3:7).
Figure 3
Figure 3. In vitro F-actin and microtubule sedimentation assay. (a) Filamentous (F-) actin (3 μM as a monomer) was precipitated by ultracentrifugation after treatment with aplyronine analogues. (b) Tubulin (3 μM as a heterodimer) was polymerized with paclitaxel (6 μM) in the presence of actin (3 μM) and/or aplyronine analogues and then precipitated by ultracentrifugation. Proteins in the supernatant (S) and the precipitate (P) were analyzed by SDS-PAGE and detected with CBB stain.
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Figure 4
Figure 4. Molecular modeling studies of the synthetic analogues of aplyronines. Conformational searches were performed as mentioned in Figure 2. (a–d) The most stable (green) and 4th stable (cyan, ΔE +11.9 kJ/mol) conformers of ApA analogue 5. Selected atom numbers are shown in blue. Two amino acid residues (Asp25 or Glu334) that interact with 5 in (a,c) are shown as sphere models in (b,d), respectively. (e,f) The most stable conformers of bis-TMSer ester analogues 7 (orange) and 8 (magenta) on actin. (g) The actin–ApA complex viewed from the bottom of the macrolide moiety. The Arg147 residue that interacts with the C13–OMe group is shown as a sphere model.
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  Aplyronine A is a macrolide isolated from Aplysia kurodai. By monitoring fluorescent intensity of pyrenyl-actin, it was found that aplyronine A inhibited both the velocity and the degree of actin polymn. Aplyronine A also quickly depolymd. F-actin. The kinetics of depolymn. suggest that aplyronine A severs F-actin. The relation between the concn. of total actin and F-actin at different concns. of aplyronine A suggests that aplyronine A forms a 1:1 complex with G-actin. From these results, it is concluded that aplyronine A inhibits actin polymn. and depolymerizes F-actin by nibbling. Comparison of the chem. structure of aplyronine A and another actin-depolymg. macrolide, mycalolide B, suggests that the side-chain but not the macrolide ring of aplyronine A may account for its actin binding and severing activity.
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  Yoneda, Kozo; Hu, Yaping; Kita, Masaki; Kigoshi, Hideo
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  Pyrene-conjugated compds. are detected by label-assisted laser desorption/ionization mass spectrometry (LA-LDI MS) without matrixes. We found that 6-amidopyrene derivs. were highly detectable by the LDI MS instrument equipped with a 355 nm laser. In a certain case of a 6-amidopyrene deriv., a mol. ion peak [M]+· and a characteristic fragment ion peak [M-42]+· were detected in an amt. of only 10 fmol. The latter peak, corresponding to the 6-aminopyrene fragment, might be generated in situ by the removal of ketene (CH2=C=O) from the parent mol. A photoaffinity amidopyrene deriv. of an antitumor macrolide aplyronine A (ApA-PaP) was synthesized, which showed potent cytotoxicity and actin-depolymg. activity. In an LDI MS anal. of the MeOH- and water-adducts of ApA-PaP, oxime N-O bonds as well as amidopyrene N-acetyl moieties were preferentially cleaved, and their internal structures were confirmed by MS/MS anal. Amidopyrene moiety might enhance fragmentation and stabilize the cleaved fragments by intramol. or intermol. weak interactions including hydrogen bonding. Our chem. probe methods might contribute to a detailed anal. of binding modes between various ligands and target biomacromols. that include multiple and weak interactions.
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  Klenchin, V. A.; King, R.; Tanaka, J.; Marriott, G.; Rayment, I. Structural basis of swinholide A binding to actin. Chem. Biol. 2005, 12, 287– 291, DOI: 10.1016/j.chembiol.2005.02.011
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  Structural basis of swinholide A binding to actin
  Klenchin, Vadim A.; King, Ryan; Tanaka, Junichi; Marriott, Gerard; Rayment, Ivan
  Chemistry & Biology (2005), 12 (3), 287-291CODEN: CBOLE2; ISSN:1074-5521. (Cell Press)
  Marine toxins targeting the actin cytoskeleton represent a new and promising class of anti-cancer compds. Here we present a 2.0 Å resoln. structure of swinholide A, a marine macrolide, bound to two actin mols. The structure demonstrates that the actin dimer in the complex does not represent a physiol. relevant entity, for the two actin mols. do not interact with each other. The swinholide A actin binding site is the same as that targeted by toxins of the trisoxazole family and numerous actin binding proteins, highlighting the importance of this site in actin polymn. The obsd. structure reveals the mechanism of action of swinholide A and provides a structural framework about which to design new agents directed at the cytoskeleton.
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  Bioconjugate Chemistry (2006), 17 (2), 524-529CODEN: BCCHES; ISSN:1043-1802. (American Chemical Society)
  The interaction between actin and aplyronine A, a potent antitumor and actin-depolymg. substance of marine origin, was investigated by photoaffinity labeling expts. Photoaffinity probes consisting of a side-chain portion of aplyronine A as a ligand, a diazirine moiety as a photoaffinity group, and a fluorophore as a detecting group were synthesized. Photolabeling expts. between actin and the probe were carried out. Actin was successfully photolabeled by the fluorescent probe and visualized clearly. The present results provide the first chem. evidence for the direct interaction between actin and the side-chain portion of aplyronine A.
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  The stable 10-I-4 species 1-hydroxy-1,3-dihydro-3,3-bis(trifluoromethyl)-1,2-benziodoxole 1-oxide (I) is the ring-closed form of o-iodoxyhexafluorocumyl alc. It is prepd. by the oxidn. of chloroiodinane II with KBrO3 in aq. H2SO4. The x-ray crystal structure of the tetrabutylammonium salt of I showed the unusual feature of an apical, neg. charged oxide ligand. 1,1,1-Triacetoxy-1,1-dihydro-1,2-benziodoxol-3(1H)-one (III) (Dess-Martin Periodinane), derived from the 1-hydroxy-1,2-benziodoxol-3(1H)-one 1-oxide by treatment with Ac2O, is an extremely useful reagent for the conversion of primary and secondary alcs. to aldehydes and ketones at 25 °C. It does not oxidize aldehydes to carboxylic acids under these conditions. It selectively oxidizes alcs. in the presence of furan rings or sulfides and does not react with vinyl ethers. Geraniol is oxidized to geranial without isomerization to nerol. Benzylic or allylic alcs. are selectively oxidized in the presence of satd. alkanols. The alc. oxidn. mechanism is discussed.
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  A new generalized Born model for estg. the free energy of hydration is presented. The new generalized Born/vol. integral (GB/VI) ests. the free energy of hydration as a classical electrostatic energy plus a cavitation energy that is not based upon at. surface area (SA) used in GB/SA hydration models but on a VI London dispersion energy estd. from quantities already calcd. in the classical electrostatic energy. The (relatively few) GB/VI model parameters are fitted to exptl. data, and parameterizations for two different at. partial charge models are presented. Comparison of the calcd. and exptl. free energies of hydration for 560 small mols. (both neutral and charged) shows good agreement (r2 = 0.94).
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  We present a method for conformational search of complex mol. systems such as macrocycles and protein loops. The method is based on perturbing an existing conformation along a mol. dynamics trajectory using initial at. velocities with kinetic energy concd. on the low-frequency vibrational modes, followed by energy minimization. A novel Chebyshev polynomial filter is used to heavily dampen the high-frequency components of a randomly generated Maxwell-Boltzmann velocity vector. The method is very efficient, even for large systems; it is straightforward to implement and requires only std. force-field energy and gradient evaluations. The results of several computational expts. suggest that the method is capable of efficiently sampling low-strain energy conformations of complex systems with nontrivial nonbonded interaction networks.
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Synthesis and Biological Activities of Aplyronine A Analogues toward the Development of Antitumor Protein–Protein Interaction Inducers between Actin and Tubulin: Conjugation of the C1–C9 Macrolactone Part and the C24–C34 Side Chain

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Abstract

Introduction

Figure 1

Results and Discussion

Figure 2

Scheme 1

Scheme 2

Scheme 3

Figure 3

Figure 4

Conclusions

Experimental Sections

General Information

Spectroscopic Analysis

Cell Culture and Cytotoxicity Assay

In Vitro F-Actin Sedimentation Assay(9a,28)

Microtubule Sedimentation Assay(8)

Molecular Modeling Studies

Synthesis and Spectroscopic Data of ApA Analogues

Thioether 9a

Phenyltetrazole Sulfone 10

Olefin 11a

Diol 11b

TBDPS Ether 12

Secondary Alcohol 12a

DMAla Ester 12b

Hemiacetal 12c

Diol 13

Trityl Ether 13a

Acetate 14

Primary Alcohol 14a

Aldehyde 15

Enamide 15a

C23–C34 Segment 16

Acetonide 17b

Aldehyde 17c

Conjugated Ester 18

Diol 18a

TBDPS Ether 19

MTM Ether 19a

Carboxylic Acid 20

TBS Ether 19b

Carboxylic Acid 21

Conjugated Ester 22a

Carboxylic Acid 23

Ester 20a

Secondary Alcohol 24

TMSer Ester 24a

ApA Analogue 5

ApC Analogue 6

Ester 21a

Bis-TMSer Ester 7

Ester 23a

Triol 25

Bis-TMSer Ester 8

Supporting Information

Terms & Conditions

Author Information

Acknowledgments

References

Cited By

Abstract

Figure 1

Figure 2

Scheme 1

Scheme 2

Scheme 3

Figure 3

Figure 4

References

Supporting Information

Supporting Information

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STEP 1:

STEP 2: