VII. Supporting appendix
30. Synthesis and early investigation of Mitsunobu MCO with a didepsipeptide monomer
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byproduct is common70,71,72 in Mitsunobu reactions, especially in the presence of excess coupling reagents, which are generally required for a macrolactonization to occur. However, an ion template would allow the linear chain to reside in a conformation that increases the rate of cyclization relative to acylation.
Collectively, these additive effects provide a range of control that either enhances the formation of one macrocycle size relative to others, or enhances diversity of oligomer size in a single reaction. In terms of concise preparations of (-)-verticilide, both approaches result in the same 24-membered N-H macrocycle (53), which upon N-permethylation provides the natural product. Contrasting approaches, the shorter preparation of (-)-verticilide (6 steps, 15% yield) using didepsipeptide 74 results in several cyclooligomers; use of tetradepsipeptide 70 and a salt effect that favors dimerization/macrocyclization to 53 is only slightly longer (8 steps, 36% yield), but is more efficient when targeting a specific natural product.
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cyclization of diastereomers 77 and 74 were apparent in salt-free MCO conditions. First, direct cyclization to the 6-membered diketomorpholine product (78) was dominant (38% yield) in the additive-free MCO, as opposed to the formation of larger oligomers with 74. In general, the smaller ring sizes were most favored, forming the 18-membered macrocycle (79) in 14% yield, and the 24-membered macrocycle (80) in 11% yield. Added salts again modified both size-distribution and efficiency of macrocyclooligomerization. NaBF4 enhanced the production of the 18- and 24- membered rings at the expense of direct cyclization to the 6-membered ring. Interestingly, KBF4, which significantly increased product formation with monomer 74, seemed to decrease the overall yield of macrocycles produced with monomer 77. The effect of CsCl was also notable, as it produced a rather even distribution of the original three ring sizes, plus a 30-membered macrocycle (81) which was not observed in salt-free conditions, in a combined isolated yield of 93%.
2.6 Examination of structural effects with the total synthesis of (-)-bassianolide From these studies, we have established a methodology to rapidly synthesize collections of CODs with unprecedented ease, and we have found that the relative distributions of products in each collection can be modulated in the presence of an alkali metal salt. This method was successfully used to synthesize the natural product (-)-verticilide, and several stereochemically and ring size-diverse analogs. However, the monomers studied up to this point were composed of alternating α-hydroxyheptanoic acid and alanine building blocks. We were interested in investigating the level of generality that might be inherent to the method. Confident that the Mitsunobu MCO could be used to synthesize any 24-membered COD natural product, attention turned to the most sterically hindered of them all, (-)-bassianolide (67), to test the generality of our method.
Figure 15. MCO of didepsipeptide 77: effect of salt additives on size distribution of products
(-)-Bassianolide (67) was first isolated from an entomopathogenic fungus, Beauveria bassiana, found on the corpses of silk worms by Suzuki and coworkers73 in the course of screening for insecticidal metabolites. (-)-Bassianolide is a 24-membered depsipeptide that is lethal to silkworm larvae at a dosage greater than 8 ppm.74 At a lower dose, this depsipeptide was found to cause atony, a symptom of muscular relaxation. Additionally, (-)-bassianolide was screened for ability to inhibit acetylcholine (ACh)-induced muscle contractions and was found to inhibit smooth muscle contractions in guinea pig intestinal cells at a concentration of 10 μM.75 Its mechanism of inhibition was found to be different than known ACh-induced muscle contraction inhibitors, papaverine (83) and verapamil (84), in that does not appear to involve changes in the binding activity of ACh to the muscarinic receptor, the membrane potential, or the contractile machinery of the intestinal wall. Because (-)-bassianolide did not change the permeability of mono or divalent cations through cell membranes during the membrane potential experiments, (-)-bassianolide most likely does not have any ionophoric properties in vivo, unlike its 18-membered analog enniantin C (82).55
Suzuki and coworkers synthesized (-)-bassianolide76 and elucidated its structure73 in 1977.
Acid hydrolysis of the natural product and comparison with an authentic amino acid sample identified N-methyl-L-leucine as the only amino acid residue, and an ether extract of the hydrolysate yielded solely α-hydroxyisovaleric acid. The stereochemistry of the hydroxy acid was identified by reducing (-)-bassianolide with LiBH4 to didepsipeptide 85, and comparing its Rf, 1H NMR, and 13C NMR spectra to synthetic didepsipeptides 85a and 85b (Scheme 31). The didepsipeptide from the natural product (85) was identical to 85a, thus the acid in the natural product was identified as D-α-hydroxyisovaleric acid. 1H NMR analysis revealed five N-methyl signals, but FD- and EI mass spectra indicated a molecular formula that was in accordance with
73 Suzuki, A.; Kanaoka, M.; Isogai, A.; Tamura, S.; Murakoshi, S.; Ichinoe, M. Tetrahedron Lett. 1977, 18, 2167..
74 Kanaoka, M.; Isogai, A.; Murakoshi, S.; Ichinoe, M.; Suzuki, A.; Tamura, S. Agric. Biol. Chem. 1978, 42, 629.
75 Nakajyo, S.; Shimizu, K.; Kometani, A.; Suzuki, A.; Ozaki, H.; Urakawa, N. Jpn. J. Pharmacol. 1983, 33, 573..
76 Kanaoka, M.; Isogai, A.; Suzuki, A. Tetrahedron Lett. 1977, 18, 4049.
Figure 16. (-)-Bassianolide’s structure leads to unique biological activity
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four N-methyl amino acid and four hydroxy acid residues, so the cyclooctadepsipeptide and cyclodecadepsipeptide were synthesized to confirm the structure.
As shown in Scheme 32, the chiral D-α-hydroxy ester starting material was synthesized in three steps from D-valine.77 In this sequence, D-valine was diazotized in the presence of sodium acetate and acetic acid to afford the α-acetoxy acid (86) with retention of stereochemistry. Then, tert-butyl ester formation was followed by deacetylation to afford the D-α-hydroxyisovaleric ester starting material (87).
The D-ester was then coupled to N-Cbz-N-methyl-L-leucine using PCl5 to form a depsipeptide species, which was then subjected to a series of convergent deprotections, traditional peptide couplings, and a final macrolactamization using the same acid chloride method (Scheme 33). With this method, Suzuki and coworkers were able to synthesize (-)-bassianolide in a 10-step longest linear sequence and 9% yield over the last 7 steps.78 The synthetic cyclooctadepsipeptide data was in agreement with that of the natural product, thus successfully verifying its structure.
77 Plattner, P. A.; Vogler, K.; Studer, R. O.; Quitt, P.; Keller-Schierlein, W. Experientia 1963, 19, 71.
78 Yields to make the hydroxy acid and amino acid starting materials were not reported.
Scheme 31. (-)-Bassianolide structure elucidation