Patterns between monomer size and salt effects explained by ITC measurements

III. Evidence for ion-templation during Mitsunobu cyclooligomerization

3.3 Correlation of ITC Measurements with MCO salt effects

3.3.1 Patterns between monomer size and salt effects explained by ITC measurements

57 Here, [AT] and [A0] are the

respective concentrations of a given product formed with and without a template, and [Amax] represents the maximum possible concentration of that product, based on the amount of available monomers. In order to account for experimental variance, the literature- accepted value for a significant template- induced change is when |AFn| > 0.09.¹¹⁶ Because this parameter has never been used for a kinetic templating reaction, it was necessary to verify that the variance within control reactions was less than 0.09, or adjust the value for a significant change in library composition accordingly.

Figure 22 outlines an example calculation for the collection of macrocycles resulting from untemplated

MCO runs from didepsipeptide monomer 74. Based on these results, the AFn within untemplated control experiments is less than 0.09 across the board, averaging out to be 0.02. This calculation was also done within multiple runs of templated MCO, and as expected, there was little to no variation between repeated runs. Therefore, |AFn| > 0.09 represents a template-induced change more than double what is observed within repeated sets of identical experiments, and is therefore able to represent the cutoff value between experimental error and a significant template-induced change for the MCO experiments. Amplification factor analysis allows us to have quantitative grounds to determine which macrocyclic products are significantly increased, decreased, or not affected in the presence of a salt added to an MCO reaction. The AFn values for all collections of macrocycles discussed later in this chapter have been calculated, and are referenced as a guideline to determine if the yield of a product has changed beyond the margin of experimental error in the presence of an ion template. With these calculations in hand, we are now able to correlate significant template effects with thermodynamic parameters obtained from ITC measurements.

58

templates. Under salt-free conditions, tetradepsipeptide 70 inherently produces cyclodimer 53 as the major product, along with 36-membered ring 72 and 60-membered ring 73 in minor amounts.

However, in the presence of NaBF4, 53 is formed exclusively. As expected, 53 strongly and favorably binds Na⁺ in a 1:1 stoichiometry. However, 72 and 73 also bind Na⁺, but in 2:1 ion to macrocycle ratios. The selectivity observed in the reaction despite the measured Na⁺ binding ability of the other two potential products can be explained by a positive templating effect:^103,99 Na⁺ promotes the preorganization^62,92 of two tetradepsipeptide building blocks (72), driven by favorable binding to the linear precursor to 53 in a conformation that accelerates an inherently favorable ring-closure.

Additionally, a combination of the inherently slow oligomerization of 70 and preorganization of a two-Na⁺ ion cyclization template, can explain why cyclotrimerization to provide 72 is much slower than cyclodimerization to form 53. Similar logic can be applied to arrive at a reason why didepsipeptide 74 did not provide the same results with Na⁺: the rate of intermolecular (Mitsunobu) coupling of 74 is much faster than that for 70, so although the yield of the 24-membered ring (53) is greater than observed in salt-free conditions, the concentrated reaction conditions and more reactive monomer (74) renders intermolecular coupling a more competitive process than in the former example. Additionally, 18-membered ring 75 and 30- membered ring 76 were found to exist in an equilibrium of 1:2 Na⁺ to macrocycle aggregates and 2:1 Na⁺ to macrocycle complexes, averaging 1.5 bound ions.^119,120 The observation of a 1:2 binding

119 Both macrocycles gave atypical sigmoidal ITC thermograms with inflection points at 0.5 and 1.5.

120 Darby, S. J.; Platts, L.; Daniel, M. S.; Cowieson, A. J.; Falconer, R. J. J. Therm. Anal. Calorim. 2016, 127, 1201.

Figure 23. Correlation of ITC data with salt effects on collections of macrocycles formed using 70^a

template 24-mem ring (53) 36-mem ring (72) 60-mem ring (73) Ka (M^-1) ΔH N Ka (M^-1) ΔH N Ka (M^-1) ΔH N Na⁺ 3.18x10⁴ − 1:1 1.56x10⁴ − 2:1 7.04x10³ + 2:1

K⁺ 2.67x10³ − 1:1 2.06x10⁴ − 1:1 2.97x10³ + 3:1 Cs⁺ <1.00x10³ 5.95x10³ − 1:1 4.27x10³ + 3:1

aITC binding affinities <1x10³ M^-1are not detected

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pattern with 75 can imply that Na⁺ promotes the preorganization of two linear-precursors, which then rapidly undergo cyclodimerization to the 36-membered ring (72) with the help of a second Na⁺ ion. Because 76 is substantially larger than 75, formation of a 1:2 metal-linear peptide complex is more likely to result in stable aggregates, as opposed to the precursor of a cyclodimerization reaction. Thus, the ITC binding pattern observed with 75, which has the strongest 2-Na⁺ binding constant, can imply that cyclization of the linear precursor is slowed by the formation of aggregates, but is then accelerated when the complex gains an additional Na⁺ 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 decreased. The 36-membered ring has the greatest enthalpically favorable 1:1 binding to K⁺, but is not produced selectively, unlike what was observed with the 24-membered ring and Na⁺. This can be explained by the overall efficacy of K⁺ as a cyclization template. An effective template must have the capacity to bind early linear intermediates in the oligomerization reaction, but also prevent them from competitively cyclizing.^99,103 In this case, the untemplated cyclodimerization reaction is very fast, and addition of K⁺ slows it, but not enough to selectively favor formation of the cyclotrimer (72). However, addition of K⁺ to the MCO with didepsipeptide monomer 74 produces the 36-membered ring in nearly double its original amount. In conjunction with the inherent fast intermolecular oligomerization of monomer 1, and strong binding to K⁺, the rate of 36-membered ring cyclization can be selectively increased. ITC indicates that the other ring sizes either weakly

Figure 24. Correlation of ITC data with salt effects on collections of macrocycles formed using 74^a

template 18-mem ring (75) 24-mem ring (53) 30-mem ring (76) 36-mem ring (72) Ka (M^-1) ΔH N Ka (M^-1) ΔH N Ka (M^-1) ΔH N Ka (M^-1) ΔH N Na⁺ 8.33x10⁴ −

1:2 then

2:1

3.18x10⁴ − 1:1 3.44x10⁴ − 1:2 then

2:1

1.56x10⁴ − 2:1

K⁺ <1x10³ 2.67x10³ − 1:1 <1x10³ 2.06x10⁴ − 1:1

Cs⁺ <1x10³ <1x10³ <1x10³ 5.95x10³ − 1:1

aITC binding affinities <1x10³ M^-1are not detected

60

bind (Ka < 1x10⁴ M^-1),¹¹⁸ or do not bind K⁺ at detectable levels (Ka < 1x10³ M^-1), so their cyclization rates and respective yields are minimally affected.

The largest cation (Cs⁺) with tetradepsipeptide 70 significantly amplified both of the larger ring sizes relative to the control, but did not favor them substantially as observed with 53 and Na⁺. ITC data shows that 72 and 73 have similar, but weak, binding affinities to Cs⁺ (Figure 23).

However, Cs⁺ with didepsipeptide monomer 74 does not result in significant amplification of macrocycles relative to the salt-free control. In these two examples, the modest change in size distribution can largely be attributed to the lack of a driving force from formation of a strong Cs⁺- binding product.