Replication of a synthetic oligomer using chameleon base-pairs

Salt bridges were used to attach polymerisable amidine monomers to an oligomeric benzoic acid template. CuAAC oligomerisation reactions in the presence of a benzoic acid 3-mer template gave the amidine 3-mer copy as the major product. Cleavage of ester linkers was used to hydrolyse off the amidine recognition units and convert the product into a benzoic acid 3-mer copy of the original template.


General details
Molecular mechanics calculations were performed using MacroModel version 13.1.141, (Release 2022-1, Schrödinger Inc.). S3 All structures were minimized first and the minimized structures were then used as the starting molecular structures for all MacroModel conformational searches. The force field used was MMFFs as implemented in this software (CHCl3 solvation). The charges were defined by the force field library and no cut off were used for non-covalent interaction. A Polak-Ribiere Conjugate Gradient (PRCG) was used, and each structure was subjected to 10000 iterations. The minima converged on a gradient with a threshold of 0.01. Conformational search was performed from previously minimized structures using 10000 steps. Images were created using PyMol. S4 Calculations were performed on simplified oligomer duplexes in which the capping groups were simplified to methyl and phenyl in order to reduce the computational cost. Amidine and carboxylates were treated as charges species and the salt bridges were fixed by constraining the distance between the nitrogens and oxygens to 1.5 ± 0.5 Å. The calculation outcomes for each duplex were sorted by energy and the 25 lowest-energy conformations were analysed.

Estimation of the ring strain for the ZIP reaction
The ring strain for the ZIP reaction was estimated by using the strain energy of the product duplexes (Estrain). The disconnection of the triazole rings into the corresponding azide and alkyne was not used due to problems in the parameterisation of azide moieties in the MMFFs force-field. The method used for disconnecting the macrocyclic structures of the duplexes through the phenyl-triazole bonds is illustrated in Figure S1. This hypothetical transformation provides a method for calculating EBond as the energy difference between two identical fragments and the connected oligomer backbone (equation S1). EBond = Eproduct -(2 x Efragment) = 467.1 -(2 x 187.0) = 93.0 kJ·mol -1 (eq. S1) Figure S1 Model system used to calculate bond connection energy EBond and lowest energy conformations from conformational searches using molecular mechanics (MMFFs force-field implemented in Macromodel with CHCl3 solvation). S3,S4 The corresponding disconnection for the antiparallel and parallel dimer duplexes is illustrated in Figure S2. The energy contribution associated with phenyl-triazole bond connection (Ebond) was subtracted from the difference between the energy of the product duplex (Eduplex) and the energy of the pre-ZIP intermediate (EpreZIP) (equation S2).
Bond product Bond fragment S19 Estrain = Eduplex -EpreZIP -n Ebond (eq. S2) where n is the number of triazole rings formed in the ZIP reaction.
The resulting ring strain is: The ring strain for the antiparallel and parallel trimer duplexes was calculated in the same way using the disconnections illustrated in Figure S3.

S20
The calculated ring strain associated with macrocyclisation in the ZIP step is 21-43 kJ·mol -1 per ring, which is in the order of the ring strain of common 5-and 6-membered rings. S5-S8 These results suggest that the ring strain associated with macrocyclisation in the ZIP step is small using this backbone.

Binding studies
The binding of amidine 6 to carboxylic acids under the suitable conditions for the templating experiment (mM concentration in a non-polar solvent) was assessed using 1 H NMR in a Bruker 500 MHz Avance III Smart Probe spectrometer. Both the free amidine monomer 6 and the salt 6·HCl were analysed. From a 1 mL solution of monomer in CDCl3 ([6·HCl]: 3.86 mM; [6]: 3.94 mM), 600 µL was added to an NMR tube. Increasing known volumes (5 µL, 5 µL, 5 µL and 20 µL) of benzoic acid were added from a 117.1 mM stock solution in CDCl3 and the spectrum recorded after each addition. The chemical shifts of the 6·HCl and 6 were monitored as a function of its concentration, in particular the amidine NH and methyl groups of the isopropyl chains. In 6, the broad signal of the methyl groups sharpens upon addition of the guest and subsequent formation of the salt bridge. For 6·HCl, these groups are already sharp without guest and no changes in chemical shift occur upon addition of benzoic acid. Figure S4 shows the 1 H NMR spectra and the plot of the change in chemical shift as a function of concentration for 6·HCl and 6. Although the concentration of guest is too high for accurate fitting of the data, the obtained results suggest that the binding constant of amidine 6 and benzoic acid in CDCl3 is bigger than 10 4 M -1 , in agreement with literature data, S9-S11 while there is no interaction when the amidine is in a salt form. Figure S4. 500 MHz 1 H NMR spectra of the amidine NH and methyl groups of the isopropyl chains of the amidine in 6 and 6·HCl for the titration of benzoic acid into 6 (3.9 mM) and 6·HCl (3.9 mM) at 298 K in CDCl3, along with the plot of the change in chemical shift of the methyl 1 H signal as a function of guest vs host ratio.

General details
The template-directed oligomerization of 6 is shown in Scheme S1. From freshly prepared stock solutions of template (in dry THF), 6 (in dry CH2Cl2) and 4-tert-butylbenzylazide (in CH2Cl2, template (1 eq.) and 6 (3 eq.) were mixed in a 1.75 mL vial containing a magnetic stirrer. The solvent was evaporated under N2 stream and dry CH2Cl2 was added. To this solution, 4-tert-butylbenzylazide was added, followed by a premixed solution of Cu(CH3CN)4PF6 (6 eq.) and TBTA (6 eq.) in dry CH2Cl2 (10 µL). The vial was flushed briefly with N2, sealed and left stirring at room temperature for 2 days. Two aliquots were taken after this time, one for UPLC analysis and the second for cleavage. Both aliquots were evaporated to remove volatile 4-tert-butylbenzylazide. One of them was dissolved in CH3CN/H20 2:1 and analyzed by UPLC. The second was dissolved in THF:H2O (3:1, 1mL) and two drops of 1M LiOH aq. soln. were then added. The solution was left to react for 1h and then analyzed by UPLC. The same procedure was followed for the non-templated oligomerization shown in Scheme S2, but no template was added. Scheme S1. CuAAC templated oligomerization of 6. Scheme S2. CuAAC non-templated oligomerization of 6.

Template concentration dependency on templating
Initially, the effect of the concentration of template and monomer 6 in the templating process was analysed. For that purpose, three different conditions were tested with [template] of 0.01, 0.1 and 1 mM and 3 equivalents of 6 in each case ( Figure S5). In all the cases, the concentration of capping azide (4-tert-butylbenzylazide) used was 1 mM. Figure S6 shows the MS spectra for the template and obtained oligomers. Figure S7 shows the UPLC S23 traces corresponding to the hydrolysis of the oligomerization crude reaction mixtures while MS spectra of the obtained species is provided in Figure S8. Figure S5. Control and templating experiments highlighted in Scheme S1 at 0.01, 0.1 and 1 mM of template with [tert-butylbenzylazide] of 1 mM, 3 eq. of 6 and 6 eq. of Cu-TBTA. In the control experiment, only 1-mer 6, capping azide and Cu-TBTA were used. S24 Figure S7. Hydrolysis of the oligomerization crude reaction mixtures shown in Figure S5.

S25
The output of the templated and control oligomerization reactions was assessed by UPLC. The ratio of the areas at 254 nm for the 2-mer (off-template pathway) over the 3-mer (templated) oligomer was calculated in each case (χ = 2-mer/3-mer). The ratio of these vales for the templating (χtemp) over the control (χcontrol) experiment (χtemp / χcontrol) was plotted as a function of the concentration of template ( Figure S9). Optimal templating is obtained when a 0.01 and 0.1 mM concentration of template is used, while more concentrated reaction leads to a loss in the template effect as the off-template pathway is favoured (more off-template 2-mer oligomer and less templated 3-mer oligomer are formed).

Solvent dependency on templating
Two different solvents were screened in order to find the most suitable one for promoting the templating reaction. Amidine-carboxylate has been reported to be stable over a wide range of non-polar solvents. S9-S11 However, in this case, the change of CH2Cl2 for THF led to a complete loss of the template effect. As shown in Figure S10, when THF is used, both control and templating experiment UPLC traces are similar, with no preferential formation of 3-mer oligomer when the template is present in the reaction.

Cap concentration dependency on templating
We have previously reported the relevance of the cap concentration on the success of covalent template-directed synthesis of linear oligomer. S12,S13 We screened the concentration of cap from 0 to 1 mM, using the best conditions from previous experiments (0.1 mM of template, 0.3 mM of 6 in CH2Cl2) in order to find the optimal cap concentration. This in turn allows the estimation of the effective molarity for the ZIP process. Figure S11 shows the UPLC traces for control and templating experiments with variable cap concentration. Figure S12 shows the UPLC traces corresponding to the hydrolysis of the oligomerization crude reaction mixtures. Figure S11. Control and templating experiments highlighted in Scheme S1 with 0.1 mM of template, variable concentration of capping azide, 3 eq. of 6 and 6 eq. of Cu-TBTA. In the control experiment, only 1-mer 6, capping azide and Cu-TBTA were used. S27 Figure S12. Hydrolysis of the oligomerization crude reaction mixtures shown in Figure S11.
Again, the output of the templated and control oligomerization reactions was assessed by UPLC. χtemp / χcontrol was plotted as a function of the concentration of end-capping agent ( Figure  S13), which shows how the product distribution depends on the concentration of the capping agent. Figure S13. Plot of the ratio of 2-mer vs 3-mer oligomers between templating and control experiments as a function of the concentration of capping azide ([template] = 0.1 mM). For the estimation of the EM, only the last three data points (right graph) were fitted to χtemp / χcontrol = 0.13 [cap] + 0.20 (R 2 = 0.97).
The ratio of the rates of off-template reaction (monitored via the formation of 2-mer oligomer) and intramolecular reaction (monitored via the formation of templated 3-mer Aoff-template/Atemplating = c [cap] (eq. S3) The data in Figure S11 can be used to determine values of effective molarity (EM) for the ZIP process by extrapolating the curve to the concentration of capping agent necessary to obtain a 1:1 ratio of the two products. These concentrations were corrected for the 5-fold difference in reactivity measured for the aromatic and aliphatic azide. S12,S13 The value of EM estimated is 32 mM.