VII. Supporting appendix
45. Synthesis of tetradepsipeptide 122
Figure 34. Stacked 19F NMR comparison of macrocycles from 122
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products with the original contents of the crude reaction mixture. As observed with the 4-ClMan hydroxy acid (110), the overall yield of the untemplated MCO was not as high as observed with our (-)-verticilide tetradepsipeptide (70), likely due to A1,3 strain from the aryl ring. However, it was much higher than observed with 110 and a collection of 24-, 36-, and 48- membered rings were isolated in 63% overall yield (Figure 34).
The 24 membered ring (131) was isolated in 35% yield as a single symmetrical conformer.
The 36-membered ring however, was slightly more complex—it was isolated in 13% yield as a symmetric conformer (132), but another macrocycle (133) that had identical molecular weight (as per LCMS analysis) and eluted approximately 3 minutes before it on preparatory HPLC, was isolated in 6% yield. Further TOF and MS/MS analysis revealed that this macrocycle had a similar time of flight and MS/MS fragmentation pattern as the 36-membered ring, which indicates that it is, indeed, the same size. However, its 1H NMR spectrum (Figure 35) has 3x the amount of peaks that it should have, in addition to 3 distinct fluorine peaks by 19F NMR (Figure 34). 2D-NMR experiments were conducted on 133 to learn more about its structure: HMBC confirmed it is one continuous macrocycle, and not two interlinked. However, 2D-NOESY showed long range cross- peaks (highlighted in red) which indicate that two 4-FMan ester linkages and one L-lac ester linkage were twisted, giving rise to an asymmetric conformation (Figure 35). This macrocycle did not appear be converting to a symmetrical conformer by 1H NMR in DMSO-d6. However, further spectral comparison between 133 and 132 revealed that the central fluorine peak of 133 overlapped with the symmetrical conformer (132). Additionally, there are some peaks that are identical between the two compounds by 1H NMR. Therefore, 133 is likely a stable and separable asymmetric conformer of 132. This type of stability between conformers has not been reported for any cyclooligodepsipeptides, to date. However, there are reports of other types of depsipeptide and
Figure 35. Relevant NOE correlations for asymmetric 36-membered ring 133 in DMSO-d6
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peptide macrocycles that exist in two separable conformers.139,140,141,142 Depsipeptide macrocycle triostin A is a good example of this—its activation energy for conformational interconversion is reported to be >22 kcal/mol, which results in two stable and separable conformers.133 X-Ray crystal structures of the conformers revealed that the large energy barrier between the two conformers likely exists because a conformational change would involve breaking hydrogen bonds on each side of the molecule, and disrupting π-stacking interactions.133
Valinomycin is known to reside in a bracelet-like conformation, which is highly stabilized by H-bonds between the amide N-H’s and ester carbonyls.143 Therefore, the high energy barrier between 132 and 133 is likely due to the activation energy necessary to break a stable H-bonded network. The amounts of 132 and 133 that were isolated in the reaction, relative to what was present by crude 19F NMR indicates that 132 is not converting to 133 over time, and must be formed from a specific conformation of the linear precursor in the MCO.
The last macrocycle (134) isolated from the reaction also had an interesting asymmetric conformation (Figure 36). Its 1H NMR spectrum revealed twice the number of peaks as expected, and 19F NMR showed two very distinct fluorine resonances. None of these resonances overlapped at all with the 24-membered ring, and TOF and MS/MS analysis confirmed that this macrocycle
139 Steinmetz, H.; Gerth, K.; Jansen, R.; Schläger, N.; Dehn, R.; Reinecke, S.; Kirschning, A.; Müller, R. Angew.
Chem. Int. Ed. 2011, 50, 532.
140 Tomooka, K.; Uehara, K.; Nishikawa, R.; Suzuki, M.; Igawa, K. J. Am. Chem. Soc. 2010, 132, 9232.
141 Denekamp, C.; Gottlieb, L.; Tamiri, T.; Tsoglin, A.; Shilav, R.; Kapon, M. Org. Lett. 2005, 7, 2461.
142 Tabudravu, J. N.; Jaspars, M.; Morris, L. A.; Kettenes-van den Bosch, J. J.; Smith, N. J. Org. Chem. 2002, 67, 8593.
143 Bystrov, V. F.; Gavrilov, Y. D.; Ivanov, V. T.; Ovchinnikov, Y. A. Eur. J. Biochem. 1977, 78, 63.
Figure 36. Relevant NOE correlations for 48-membered ring 134 in DMSO-d6
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was not a conformer of the 24-membered ring, but instead our first 48-membered ring, isolated in 8% yield. HMBC confirmed that 134 was a continuous 48-membered macrocycle, and 2D- NOESY revealed that its expected C4-symmetry was broken by 2 twisted ester linkages (highlighted in red) to afford 1H NMR (Figure 36) and 19F NMR (Figure 34) spectra with apparent C2-symmetry.
Having established the structures of our new macrocycles formed without templation, tetradepsipeptide 122 was subjected to templated MCO conditions. The results of the templated experiments are summarized in Figure 37 . Overall, the collection of ring sizes seemed to be less promiscuously affected by the addition of a metal ion template. This is not terribly surprising because valinomycin’s rigid and hollow cylindrical structure is what gives it such specific potassium ion affinity. The D,L-macrocycles that we have analyzed previously have much more inherent conformational freedom to interact with a wider range of ion sizes. The D,D,L,L- stereochemical pattern allows the macrocycles in this collection to adopt more rigid, “valinomycin- like” conformations. Therefore, matching ion binding site size with cation size will be more necessary for a significant templating effect in the MCO.
In this example, 131 and 132 were the only ring sizes that were substantially affected by the presence of an alkali metal salt template. However, no significant changes were observed with Na+, which has largely been the metal cation with the most substantial effect on D,L-macrocycles.
The D,D,L,L-macrocycles appeared to be more substantially influenced by larger ion templates.
Figure 37. Examination of stereochemical effects in the MCO by synthesis of D,D,L,L-macrocycles from 122a
template 24-mem ring (131) 36-mem ring (132) Ka (M-1) ΔH N Ka (M-1) ΔH N Na+ <1.00x103 <1.00x103
K+ <1.00x103 8.85x103 − 1:1
aITC binding affinities <1x103 M-1are not detected
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Most notably, the 36-membered ring was formed in nearly 3x its original amount in the presence of K+. The largest cation (Cs+) significantly amplified both the 24- and 36-membered rings, and provided a 95% isolated yield of macrocycles overall.
As mentioned earlier in this chapter, valinomycin had the best K+/Na+ selectivity of all known potassium complexones.53 In methanol, it is reported to have a binding affinity for K+ that is >10,000x stronger than Na+. Valinomycin can also strongly bind Cs+,144 but its affinity for K+ in methanol is still 3x greater. As valinomycin’s claim-to-fame is its incredible K+/Na+ selectivity, ITC data was obtained to measure Na+ and K+ binding affinities with 131 and 132 (Figure 37).145 Neither macrocycle has affinity for Na+, and only the 36-membered ring (132) binds K+, albeit more weakly than that observed in previous examples of depsipeptides with potassium ion-affinity.
The limit of accurate detection on our ITC instrument is 1x103 M-1, so if 132 bound sodium ions with similar affinity as valinomycin (around 4x100 M-1), it would not be detected.
However, the K+-selective binding here allows the 36-membered ring (132) to be increased substantially because its rate of formation can be increased selectively over the 24-membered ring.
The 36-membered ring is not formed as the only product due to the lack of a strong driving force from formation of a strong K+-binding product. Nonetheless, this example marks the most substantially increased example of a cyclotrimerization that we have observed from a tetradepsipeptide monomer thus far.