I would also like to thank Michael Crocker for super fun NMR discussions, and for a shared appreciation of cats and unicorns. I especially want to thank Jade Bing, who has been a voice of reason in the lab (and out of the lab), close friend and roommate—thanks for all the feedback on my presentations, for tolerating my cat when he steals your food, and to many fun times outside the lab.
Introduction to cyclic peptides and depsipeptides
The high prevalence of macrocycles among these molecules, exceeding the rule of five, suggests that their cyclic structure is important for favorable pharmaceutical properties. The relationship between membrane structure and permeability is an important determinant of whether a target exceeding the rule of 5 will be orally bioavailable.
Chemical synthesis of N-methyl peptides and depsipeptides
Traditional methods
However, SPPS is considerably more difficult than solution-phase peptide synthesis in the case of N-methyl-depsipeptide synthesis.13 This is due to further reduced reactivity of resin-bound N-methylamine nucleophiles towards the active ester species in addition to low yield ester. -formation reactions.
UmAS as a new method to prepare depsipeptides
A reaction that should have resulted in a single diastereomer instead produced a 4:1 ratio of diastereomers, due to epimerization of the α-hydroxy carbon (Scheme 9). Leighty and Shen also found that MOM protection of the crude alcohol prevented their retro-Henry decomposition.
Isolation and prior synthesis of (-)-verticilide
The final steps in Ōmura's synthesis of (−)-verticilide (Scheme 13) involve a series of convergent deprotections and standard peptide couplings, the last being a ring-closing macrolactamization. First-generation efforts on an UmAS-based approach to (-)-verticilide Ōmura's synthesis26 provide an example of an excellent modern route to.
First generation endeavors toward an UmAS-based approach to (-)-verticilide
This product was not observed in the crude NMR of Henry, so it was assumed to be formed during the MOM protection step. The yield of the initial experiment was still low, but the reaction was much cleaner.
UmAS-based approach to (-)-verticilide: revised route
However, the other 26 hexapeptide scaffolds gave either a mixture of products or no reaction due to the conformational nature of the cyclic peptide. It is assumed that N-methylation did not occur in these reactions due to the conformation of 53 in solution (Figure 8).
Conclusion
Synthesis of size-diverse collections of cyclodepsipeptides via an ion-templated
Background and importance of macrocycle size
On average, macrocyclic ligands were found to occupy significantly more 'hot spots' than small molecule ligands (5.2 vs 3.6),6 further reinforcing their importance in drug discovery.
Cyclooligomerization: a rapid method to synthesize complex macrocycles
In both examples, the focus was on optimizing overall yield and size diversity, as opposed to targeting a specific ring size. Both groups faced difficulties with the formation of insoluble linear polymers as opposed to cyclized products.
Initial discovery of a Mitsunobu cyclooligomerization (MCO)
At the most dilute concentration (0.5 mM), little overall reactivity or selectivity was observed, affording the 24-membered ring (53) and 36-membered ring (72) in 18 and 21% yields, respectively. In the most concentrated reaction conditions, more polymerized linear chains were observed, thereby confirming that more dilute conditions were required for efficient cyclization.
Ion-templated Mitsunobu cyclooligomerization
However, NaBF4 remained inert in the reaction and promoted the exclusive formation of the 24-membered ring. Here, an ion template would allow the linear chain to remain in a conformation that increases the rate of cyclization to the 24-membered ring compared to continuous oligomerization.
Examination of stereochemical effects
First, direct cyclization to the 6-membered diketomorpholine product (78) predominated (38% yield) in additive-free MCO, in contrast to the formation of larger oligomers with 74. NaBF4 enhanced the production of 18- and 24-membered rings at the expense of direct cyclization into a 6-membered ring.
Examination of structural effects with the total synthesis of (-)-bassianolide
- Introduction: (-)-bassianolide and its previous synthesis
- Overview of the proposed Mitsunobu MCO-based approach
- Development of a Mitsunobu MCO-based synthesis of (-)-bassianolide
The stereochemistry of the hydroxy acid was identified by reducing (-)-basianolide with LiBH4 to didepsipeptide 85, and comparing its Rf, 1H NMR and 13C NMR spectra with synthetic didepsipeptides 85a and 85b (Scheme ). Raising the reaction temperature only slightly increased the yield (from 30 to 43%), at the expense of enantioselection.
Conclusion
Although this synthesis is a lower yield than our original synthesis of (-)-verticillide, it is fewer steps than the Suzuki route and provides the natural product in similar yield. This synthesis also demonstrates that our route to cyclic N-methylated depsipeptides can be applied to substrates that are difficult to make in nature.
Evidence for ion-templation during Mitsunobu cyclooligomerization
- Introduction
- Classification of templated oligomerization reactions
- Isothermal titration calorimetry (ITC)
- Experimental design to determine template effects in the Mitsunobu MCO
- Analysis of macrocycle-ion binding interactions with ITC measurements
- Normalized amplification factor analysis: a measure of significance in template effect
- Correlation of ITC Measurements with MCO salt effects
- Patterns between monomer size and salt effects explained by ITC measurements
- Use of ITC measurements to explain MCO salt effects with D,D-macrocycles
- Analysis of structural and thermodynamic trends
- A test of generality: correlation of trends in binding with a new D,L monomer
- Conclusion
A key factor affecting the size distribution of the products formed in ion-patterned MCO was the size of the starting monomer material. The thermodynamically disfavored di-ion binding of the 24-membered ring may result in a decreased rate of cyclization and potentially promote the formation of a 30-membered ring, which is not formed in the unformed oligomerization.
Trends in monomer stereochemistry & sterics with MCO size distribution
- Introduction
- Correlation of stereochemistry and sterics: D,L-macrocycles
- MCO-based synthesis of (-)-bassianolide: A case study
- Comparison of steric effects with L,L-didepsipeptides
- Mandelic acid residues violate the expected C n -symmetry of D,L macrocycles
- Correlation of stereochemistry and sterics: D,D,L,L-macrocycles
- Introduction to valinomycin & synthetic derivatives
- MCO-based synthesis of 4-FMan-valinomycins
- MCO-based synthesis of the natural valinomycins
- Analysis of steric and stereochemical trends
- Conclusion
Most strikingly, the 36-ring was formed in almost three times the original amount in the presence of K+. Future studies will aim to expand the scope of the templates used in the MCO for the synthesis of D,D,L,L macrocycles.
Illuminating the dark side of natural products
Introduction
- Ryanodine receptor 2 (RyR2) as a target for disease cures
- Uncover novel biological functions with unnatural enantiomers of natural products . 85
3 μM ent-21 exhibited a dual effect on Ca2+ sparks: a significant reduction in the rate of spontaneous Ca2+ sparks (measured as spark frequency) and the amount of Ca2+. To test whether ent-21 crosses the cell membrane, we measured spontaneous Ca2+ release in intact Casq2-/- cardiomyocytes.
Conclusion
However, at this time, our report highlights a rare and exciting discovery of an unnatural enantiomer that potently suppresses RyR2-mediated Ca2+ efflux, whereas the natural product is completely inactive. In the case of verticillide, the unnatural enantiomer exerts a similar but mechanistically orthogonal pharmacological behavior to the existing Ca2+ release inhibitors flecainide and tetracaine, while the natural product is inactive.
Conclusion and outlook
Conclusion
Outlook
- Hetero-MCO for the synthesis of collections RGD-containing macrocycles
- Development of improved MCO purification
- Drug delivery depsipeptides
- Discovery of new compatible Lewis acidic templates in the MCO
Flash column chromatography (SiO2, 10-20% ethyl acetate in hexanes) afforded the pure amide as a colorless oil (18 mg, 34% yield). The residue was subjected to flash column chromatography (SiO2, 10-25% ethyl acetate in hexanes) to provide the amide as a waxy yellow solid (680 mg, 63%).
Supporting appendix
Synthesis and characterization of organic molecules
Isothermal Titration Calorimetry
The loading syringe was used to gently tap the bottom of the reference cell to release any trapped bubbles and ultimately remove any displaced methanol in the overflow area. Control experiments were performed under the same conditions by injecting the alkali metal salt solution into methanol alone.
Ion mobility and MS/MS result summary
Biological methods
Isolated ventricular myocytes were plated on laminin-coated glass coverslips and allowed to adhere for 10 min at room temperature (22–24°C). The amplitude of the caffeine-induced Ca2+ transient was used as an estimate of the total SR Ca2+ content.
NMR spectra
Enantioselective Henry temperature & bromonitromethane addition screen
UmAS with N-methyl alanine isopropyl ester halogen source screen
N-Chloramine in UmAS optimization attempts
UmAS with N-methyl alanine isopropyl ester temperature study
MOM-deprotection optimization
Tetradepsipeptide saponification attempts
N-Methylation optimization attempts: round 1
N-Methylation of ion-coordinated N-H 24-membered ring
N-Methylation conditions to afford (-)-verticilide and derivatives
Enantioselective bromonitromethane addition into isobutyraldehyde
Optimization of one-pot bromination/amidation UmAS to afford α-hydroxy amide 97
Investigation of the role of NaBF 4 in cyclodimerization to N-H bassianolide
Investigation of other chaotropes and solvents
Global deprotection attempts
FDA approved cyclic peptide and depsipeptide drugs
Depsipeptide macrocycles violate Lipinski’s rule of five
Stereochemistry and N-methylation heavily influence membrane permeability
Condensative amide synthesis vs UmAS
Conventional α-hydroxy amide synthesis vs Henry/UmAS sequence
Hypothesized conformation of the Cu(II) complex
Major products of UmAS with N-chlorovaline
Hypothesized solvent-dependent conformations of 53
Hypothesized shift of hydrogen bond network in free and ion coordinated 53
Macrocycle binding modes. 6 Figure adapted from Nat. Chem. Biol. 2014, 10, 723
Cyclooligomeric depsipeptide (COD) natural products
MCO concentration studies
MCO of tetradepsipeptide 70: effect of salt additives on size distribution of products
Effect of salt additives on size distribution of products from MCO with a didepsipeptide
MCO of didepsipeptide 77: effect of salt additives on size distribution of products
Comparison of macrolactamization yields to afford (-)-bassianolide and its N-desmethyl
Helical hydrogen bonding conformation of N-desmethyl analogs
Structural comparison of tetradepsipeptide monomers 102 and 48
Mitsunobu MCO strategy for measurement-based macrocycle synthesis
Schematic of an ITC microcalorimeter and sample ITC thermogram
Determination of variance within untemplated reactions
Correlation of ITC data with salt effects on collections of macrocycles formed using 70 a
Correlation of ITC data with salt effects on collections of macrocycles formed using 74 a
Correlation of ITC data with salt effects on collections of macrocycles formed using 77 a
Structural and thermodynamic trends for depsipeptide synthesis via ion-templated MCO
Chlorine isotopes are a helpful tool to identify new macrocyclic products
Use of ITC to predict changes in product size distribution from 110 a
HyIv-containing COD natural products
Relationship between steric hindrance and MCO product size distribution without salt
Conformation-induced symmetry-breaking effects in C 2 -symmetric depsipeptides
Steric comparison between monomers 122 and 123
Stacked 19 F NMR comparison of macrocycles from 122
Relevant NOE correlations for asymmetric 36-membered ring 133 in DMSO-d 6
Relevant NOE correlations for 48-membered ring 134 in DMSO-d 6
Examination of stereochemical effects in the MCO by synthesis of D,D,L,L-macrocycles
Comparison of MCO with HyIv-containing monomers 123 and 102
Steric and stereochemical trends for depsipeptide synthesis via Mitsunobu MCO
Overview of the relationship between new method development and therapeutic development
RyR-modulating small molecules
Biological relevance of unnatural mirror image isomers of natural products
Ca 2+ spark measurements of RyR activity in Casq2 -/- cardiomyocytes
Free Ca 2+ measurements in spark assay conditions
Enantiomer-dependent inhibition of RyR2-mediated Ca 2+ release with propafenone
Intracellular Ca 2+ handling in intact Casq2 -/- cardiomyocytes
Heart rate (HR) response in Casq2 -/- mice
Surface electrocardiogram parameters from recordings in Casq2 -/- mice
Summary of our discovery of a novel inhibitor of RyR2-mediated Ca 2+ leak
Amphiphilic polypeptide vehicle
MCO with polar side chain-containing monomer 143
Traditional solution phase coupling
Epimerization through oxazolone formation
Solid phase peptide synthesis
Diketomorpholine formation
Epimerization through ketene formation
Speculated UmAS mechanism
UmAS can be applied to depsipeptide synthesis by coupling an α-hydroxy-bromonitroalkane (12) with an amine to form α-hydroxyamide (13) intermediates.18 Alternatively, it is formed in solid-phase depsipeptide synthesis as shown in Scheme 7.
Enantioselective α-hydroxy amide preparations
This epimerization occurs due to deprotonation and reprotonation of the α-hydroxy center by the organic base used in the traditional amide coupling. UmAS mechanistically avoids epimerization of the α-hydroxy carbon and also eliminates the functional group manipulation step common to carboxylic acid intermediates.
Epimerization of the α-hydroxy carbon
To avoid the problem of epimerization, Leighty and Shen developed a new strategy using an enantioselective Henry addition and protection, followed by UmAS to prepare α-hydroxyamides (Figure 5). Leighty and Shen investigated the chemistry of copper acetate-bis(oxazoline)22 for the Henry addition, but found that these methods only yielded the desired product in a moderate enantiomeric excess.18 It is hypothesized that the copper counterion changes the structure of the substrate-bound can affect the catalyst. (Figure 6).23 By increasing the steric properties on one side of the Cu(II) complex, Leighty and Shen hypothesized that the aldehyde would have a greater chance of coordinating into the desired position of the complex.
Enantioselective synthesis of MOM-protected α-oxy bromonitroalkanes and their use in
A range of 19 different MOM-protected bromonitroalkanes ee) were then coupled to α-methylbenzylamine (19) (Scheme 10) to confirm conservation of configuration during coupling. In each case, the coupling afforded the desired amide (20) as a single diastereomer in moderate to good yield, confirming that the α-oxy center did not epimerize under UmAS.
Ōmura’s preparation of 2-hydroxyhepanoic acid benzyl ester for structure elucidation
Then, TBDMS protection of the hydroxyl group followed by transesterification and finally deprotection afforded the α-hydroxy ester (26) in excellent yield.
Preparation of R-2-hydroxyheptanoic acid benzyl ester
Final steps in Ōmura’s synthesis of (-)-verticilide
Proposed synthesis of (-)-verticilide incorporating the enantioselective Henry and UmAS
They assumed that the reaction was slow at lower temperatures, so the reaction time was increased. Since the reaction proceeded both cleanly to the carboxylic acid and the amide on heating, these conditions were used for UmAS with the N -methyl alanine ester ( Table 4 ).
New strategy to obtain the N-methyl amide: sequential UmAS and methylation steps
Since the N-H bond is more polarized than the α-C-H bond, deprotonation of the nitrogen should proceed faster. In this case, entropic strain from the N -methylamide 14 and activation of the isopropyl ester under the acidic reaction conditions raised the ground state energy of the free alcohol ( 37 ), thereby reducing the energy barrier for its intramolecular cyclization to 38 .
Ester saponification
Initial Mitsunobu conditions were taken from Martin's synthesis of macbecin I and herbimycin A33 because sterics around the alcohol in Martin's synthesis were similar to those of the desired tetradepsipeptide. Although the use of DEAD in the conditions provided the tetradepsipeptide in moderate (63%) yield, it was extremely difficult to separate DEAD byproducts from the desired product.
Tetradepsipeptide saponification conditions
After the first Mitsunobu reaction, 40 was divided into two parts to be saponified and deprotected by MOM, respectively.
Mitsunobu conditions to form the tetradepsipeptide
Single formation of 39 , 42 , and 43 confirmed that the central ester bond of 40 cleaves much faster than the isopropyl ester in the presence of lithium hydroxide and hydrogen peroxide. Under both conditions where the carboxylic acid tetradepsipeptide 41 was formed, the amount of diketomorpholine 42 appeared to increase with time by NMR.
Proposed tetradepsipeptide acid decomposition mechanism
This observation confirmed the instability of 41 because it decomposes not only under the basic reaction conditions but also in the post-treatment state. As the tetradepsipeptide carboxylic acid 41 proved too unstable to be used in the subsequent convergent Mitsunobu coupling, this proposed route to (−)-verticilide was no longer viable and was revised.
Revised route to (-)-verticilide
MOM deprotection of 45 with BF3•OEt2 and thiophenol afforded alcohol 46 in 86% yield with no observable formation of diketomorpholine by-products. If BF3•OEt2 was added to the reaction before thiophenol, a large amount of benzyl deprotection was observed.
Revised route: tetradepsipeptide formation
MOM ether 48 was deprotected with BF3•OEt2 and thiophenol to give tetradepsipeptide alcohol 50 in 86% yield without formation of diketomorpholine degradation products. In the literature, depsipeptide macrolactonization tends to exhibit low to moderate yields due to the tendency of the alcohol to eliminate and the sterically hindered nature of the carboxylic acid.
Revised route: seco-acid formation
Due to its insolubility, the conditions to benzyl deprotect 48 were adjusted to a 10:1 mixture of ethanol and dichloromethane instead of methanol, giving tetradepsipeptide acid 49 in quantitative yield. Macrolactamization is therefore generally preferred to the macrocyclization step in cyclodepsipeptide synthesis.39 For example, macrolactonization of depsipeptide FK22840 proved difficult, as most conditions yielded only recovered starting material or alcohol elimination byproducts.
Revised route: octadepsipeptide formation
In this synthesis, optimized Mitsunobu macrolactonization conditions of 52 use only 5 equivalents of DIAD and 6 equivalents of PPh3, less than half the amount used in the subsequent precedents. In addition, 52 was cyclized without inverting the stereocenter under Yamaguchi macrolactonization conditions to give nonsymmetrical macrocycle epi-53 in 51% yield.
Macrolactonization of octadepsipeptide seco-acid 52
The first example in Scheme 25 involves N-methylation of a resin-bound cyclic hexapeptide scaffold (54) to generate a diverse library of N-methylated cyclic peptides for the discovery of orally bioavailable scaffolds. 42 N-methylation conditions include treatment of the cyclic peptide scaffold with lithium tert- butoxide in THF, followed by removal of the base solution and addition of methyl iodide in DMSO to selectively afford the trimethylated product (55). MS analysis of theonalapeptolides was impossible due to all fragment ions of the same molecular mass, such as Ala and β-Ala; Leu, allo-Ile and Me-Val; Me-Leu, Me-Ile and Me-allo-Ile.
Known methods of N-methylation applied to macrocyclic peptides and depsipeptides
The last example in Scheme 25 is N-dimethylation with CT3I for receptor binding studies of the natural product PF1022A44 (58). This may indicate that the solvent has an effect on the stability of the potassium coordination complex.
Total synthesis of (-)-verticilide summary
Carrying out the reaction at 100 °C was necessary due to the sterically hindered cyclization, and a non-polar solvent (toluene, 50 mM) was required to promote oligomerization over monocyclization, ultimately isolating valinomycin in 7% yield . In addition, epimerization was observed at both chiral centers on the oxazoline under basic reaction conditions, so the opposite enantiomer of threonine was used to prepare the oxazoline to account for this.
Cyclooligomerization methods to synthesize oligomeric peptide and depsipeptide
Fairlie60 and Pattenden61 developed amidation-based cyclooligomerization strategies to access a variety of thiazole-containing peptide macrocycles for conformational studies. Pattenden was able to obtain high yields of macrocyclic products but observed only two ring sizes cyclotrimer and cyclotetramer.
Discovery of a Mitsunobu cyclooligomerization reaction
Careful analysis and purification of the reaction mixture afforded the 24-membered ring (53) in 63% and small amounts of two other cyclodepsipeptide oligomers. 1H NMR analysis of these cyclooligomers revealed very similar spectra that differed primarily in the chemical shift rather than the coupling pattern of each proton, reinforcing the expectation that these products are constitutionally identical.
Ion-templated cyclization reactions
Compared to conditions without salt additive, the addition of NaCl, KCl, or CsCl did not increase the yield of the 24-membered ring 53. In the presence of any of these three salts, the production of the 18-membered ring 75 was completely suppressed.
Synthesis and early investigation of Mitsunobu MCO with a didepsipeptide monomer
However, a shift in the size distribution of macrocyclic products was observed in the presence of NaBF4, KBF4, and CsCl. Enhancement of ring size in the presence of a salt additive was most significant for the 36-ring (72), which was isolated in 44% yield with KBF4, almost double the yield under salt-free conditions.
Suzuki’s starting material preparation
This phenomenon was not observed with the N-permethylated hexadepsipeptide ennianthin C or any of the N-desmethyl derivatives. Suzuki and co-workers also observed increasing difficulty in the cyclization of N-methylated derivatives, which was directly proportional to the amount of non-N-methylated leucine residues (Figure 17).
Suzuki's synthesis of (-)-bassianolide
Similar to Suzuki's route, our synthesis (Scheme 34) will use L-leucine as the starting material for amino acids, but an S-α-hydroxy acid surrogate will be used in place of D-α-hydroxyisovaleric acid. Based on previous selectivity and reactivity patterns with larger monomers, we predict that the tetradepsipeptide seconic acid will be able to favorably undergo cyclodimerization to the 24-ring in a Mitsunobu MCO.
Proposed MCO-based synthesis of (-)-bassianolide
Thus, we hypothesized that the Blay ligand (27) may be too bulky to effectively catalyze the addition of bromonitromethane to α-substituted aldehydes. Around the time our targeted addition of bromonitromethane was in development, Schwieter discovered that a nitroalkane could be subjected to UmAS conditions in the presence of a bromonium source to provide amide products in yields similar to the original conditions from a bromonitroalkane starting material.81 the obvious challenges faced in the synthesis of bromonitroalkane 92 , nitroalkane 95 was synthesized and a one-pot bromination/UmAS approach was instead followed to synthesize α-hydroxy amide 97 ( Scheme 35 ).
Enantioselective synthesis of a nitroalkane donor for one-pot UmAS
One factor to consider when implementing a one-pot UmAS amidation strategy (Table 11) is that the reaction is considerably more heterogeneous than the standard UmAS reaction. However, this yield was significantly lower than that observed under standard UmAS conditions using bromonitroalkane 92 (78% yield).
Oxime byproduct
Replacing the solvent with 2-Me THF resulted in poor conversion due to the insolubility of the reactants, and using NIS instead of NaI resulted in reduced yields due to reagent clumping and formation of a foreign pasta. This removed the major reaction byproducts by FCC, and the diastereomers of 97 were separated using preparatory HPLC.
Traditional UmAS approach vs one-pot UmAS approach summary
For convenience, the amine equivalents were reduced to 1.2, resulting in significantly less oxime byproduct 98 and a slightly higher yield of 97. 84 The starting nitroalkane was only 87% ee, so the minor enantiomer reacted to form the specific amount of D,L-epimer 97.
Synthesis of the tetradepsipeptide monomer en route to (-)-bassianolide
The reaction without the salt was largely homogeneous, unlike the reaction with NaBF4, which had a significant amount of white precipitate. When the amount of NaBF4 in the reaction was less or greater than five equivalents, little cyclized product was formed.
Attempted octadepsipeptide synthesis
Furthermore, synthesis of (−)-bassianolide using our first-generation approach was not possible because alcohol 103 and acid 104 were unable to undergo Mitsunobu coupling to form octadepsipeptide 105 ( Scheme 39 ). Despite its modest yield, Mitsunobu cyclooligomerization proved to be a more efficient method to synthesize 91 than Suzuki's linear macrolactamization approach.
N-Permethylation of N-H bassianolide 91
The data from ITC can be used to rationalize why different amounts of 24- and 36-membered rings are produced from tetradepsipeptide monomer 70 and didepsipeptide monomer 74 in the presence of the same ion. In the presence of a slightly larger cation, K + , with tetradepsipeptide 70, the 36-membered ring (72) is significantly increased relative to the control, while the 24-membered ring (53) is reduced.
Synthesis of a new monomer to test our hypothesis
The overall yield of the untemplated MCO with 110 was quite low (47%), probably due to the steric effect of A1,3 strain (from the 4-chloro mandelic acid residue) on the Mitsunobu reaction. Finally, 112 and 113 bind Cs+ weakly, whereas 111 does not, which is hypothesized to lead to modest enhancement of both of the former.
Investigation of steric hindrance, independently in α-oxy and carboxylic acid Mitsunobu
Therefore, we hypothesized that the use of 119 would be a more reactive substitution for 102 and potentially allow the formation of a larger ring distribution in addition to the formation of the 24-membered ring (91) in higher yield. Unfortunately, 119 did not afford any additional macrocyclic product relative to its original tetradepsipeptide congener—the monomer was mostly converted to linear acylated DIAD derivatives, and 5% yield of the 24-membered ring was observed.
MCO of mixed L,L,D,L-tetradepsipeptide 117
Interestingly, the 24-membered ring was the only ring size isolated from the reaction, just as the 24-membered ring was the only ring size formed with the tetradepsipeptide precursors of (-)-bassianolide and (-)-verticilide. This phenomenon was particularly evident with the 18-membered ring (111) - the most sterically congested of the three sizes formed from the MCO, and therefore most likely to contain a twisted aryl ring or rotated ester linkage to relieve ring strain .
Synthesis of tridepsipeptide amine 128
19F NMR could therefore be used to measure yields from crude reaction mixtures, as 1H NMR has so far not been a viable option due to overlapping peaks. Hydroxy acid 122 was then subjected to our optimized salt-free MCO conditions for tetradepsipeptide monomers and the crude reaction mixture was analyzed by 1H NMR and 19F NMR.
Synthesis of tetradepsipeptide 122
Since valinomycin is famous for its incredible K+/Na+ selectivity, ITC data were obtained to measure the Na+ and K+ binding affinities of 131 and 132 (Figure 37).145 None of the macrocycles has an affinity for Na+ and only the 36-membered ring. (132) binds K + , albeit more weakly than that observed in previous examples of depsipeptides with affinity for potassium ions. However, K + -selective binding here allows the 36-membered ring (132) to be substantially enlarged because its rate of formation can be selectively increased over the 24-membered ring.
Synthesis of tetradepsipeptide seco-acid 123
The largest cation (Cs+) significantly strengthened both the 24- and 36-membered rings, and provided a 95% isolated yield of macrocycles overall. 144 Valinomycin's binding affinity for cesium ions in methanol was reported to be about 2x104 M-1, and in the same article its affinity for potassium ions in methanol was reported to be about 6x104 M-1 (ref. 146).
MCO with 123 to synthesize montanastatin and valinomycin
Synthesis of new valinomycin-like tetradepsipeptide monomers revealed that amplification of macrocycles in the presence of a particular template can be tuned to be more selective with more conformationally rigid D,D,L,L macrocycles. Calsequestrin (CASQ) is a polymeric Ca 2+ -binding protein located in the junctional SR near the luminal opening of the RyR.
Enantioselective synthesis of COD natural products
In addition, the enantiomer of verticilide (ent-21) and both synthetic precursors were synthesized to determine whether the bioactivity was due to specific chiral receptor-ligand interactions. Thus, two verticilide synthesis approaches were used to obtain the verticilide congeners necessary to investigate activity against RyR2.
Macrocyclooligomerization (MCO) and modular convergent routes to verticilide
To get a sense of structure-activity relationship of any observed activity, we wanted to test the desmethyl macrocyclic precursor (53) to verticillide, and the linear macrocyclization precursor to 53. First, nat-(-)-verticillide was synthesized using our Mitsunobu MCO approach starting from tetradepsipeptide seco-acid 70 in an 8-step longest linear sequence.88 To obtain the linear precursor to verticillide, the synthesis of common tetradepsipeptide intermediate 48 was deduced to finally access 52 by a series of convergent step deshielding and coupling (Scheme 49).
Synthetic sequence to access ent-(+)-verticilide
Consistent with inhibition of RyR2-mediated Ca2+ release, ent-21 reduced diastolic Ca2+ levels, reduced Ca2+ transient amplitude, and delayed the time to peak of rhythm transients. To determine whether ent-21 inhibition of Ca2+ release in cardiomyocytes in vitro translates into anti-ventricular arrhythmia activity in vivo, Casq2-/- mice were injected intraperitoneally (i.p.) with 30 mg/kg of nat-21, ent-21, or a DMSO equivalent volume .
Hetero-MCO synthesis of collections of RGD macrocycles
The rigid structure of cyclic RGD-containing peptides222 has proven useful in the development of Cilengitide© and other analogues. Instead, formation of RGD-containing oligomers with mono (140) and di-incorporation (141) of 74 was identified by LCMS.226 This preliminary result is promising for our goal of developing a controlled hetero-MCO.
Preliminary RGD macrocycle synthesis results: homo-MCO vs hetero-MCO
The residue was subjected to flash column chromatography (SiO2, 10-20% ethyl acetate in hexanes) to yield the major diastereomer as a waxy yellow solid (1380 mg, 80%). The residue was subjected to flash column chromatography (SiO2, 20-45% ethyl acetate in hexanes) to yield the amide as a colorless oil (313 mg, 65%).
