Catalytic length-controlled oligomerization with synthetic programmable templates

Nature uses templated length-controlled oligomerization to process genetic information. Templates that are DNA and RNA based and fully synthetic have also been developed for preparing unnatural oligomers. However, these reactions require stoichiometric amounts of the template for product formation. Here we report a catalytic macrocyclic template that promotes the oligomerization of a small-molecule substrate with a remarkable degree of length control. The design of the template is based on rigid oligoproline moieties decorated with catalytic sites in a defined spatial arrangement. The dimension of the macrocycle and the number of catalytic moieties determine the number of monomers that are incorporated into the growing oligomer, thus allowing access to specific products with lengths preprogrammed by the template. Templated synthesis is a valuable method for the preparation of oligomers but requires stoichiometric amounts of the template. Here a catalytic macrocyclic template is reported that promotes the oligomerization of a small-molecule substrate and controls its length. The length of the oligomer correlates with the dimensions of the template.

Nature uses templated length-controlled oligomerization to process genetic information. Templates that are DNA and RNA based and fully synthetic have also been developed for preparing unnatural oligomers. However, these reactions require stoichiometric amounts of the template for product formation. Here we report a catalytic macrocyclic template that promotes the oligomerization of a small-molecule substrate with a remarkable degree of length control. The design of the template is based on rigid oligoproline moieties decorated with catalytic sites in a defined spatial arrangement. The dimension of the macrocycle and the number of catalytic moieties determine the number of monomers that are incorporated into the growing oligomer, thus allowing access to specific products with lengths preprogrammed by the template.
Templated synthesis is key to the production of natural oligomers from the respective monomeric building blocks 1 . For example, the genetic information is transcribed from DNA into RNA and then translated into peptides and proteins. The natural DNA-based oligomerization machinery has been manipulated by scientists such that it allows for the synthesis of any desired complementary DNA and RNA strand [2][3][4] . This approach has even been used for the synthesis of sequence-controlled non-natural oligomers (Fig. 1a) [5][6][7][8][9][10] . Impressive progress has also been made in templated oligomer synthesis with non-DNA-based templates 11 and has enabled access to macrocycles 12 and cages 13 from monomeric non-natural building blocks. Furthermore, dynamic covalent chemistry tools have facilitated the creation of self-replicating macrocycles [14][15][16] . These are formidable achievements because even the controlled formation of macrocycles from a single precursor is still challenging 17 . Templating also allowed for the synthesis of linear oligomers with length control, which is particularly difficult because they bear at least one reactive terminus [18][19][20][21] . The preparation of such synthetic oligomers with a defined length requires otherwise controlled polymerization conditions 22,23 or successive couplings of the monomers with experimental interventions at each step 24,25 . The templated formation of synthetic oligomers in one pot is therefore an intriguing and enabling alternative to access non-natural oligomers.
An intrinsic limitation of DNA-based templates and all other scaffolds used so far is the tight binding between the template and the complementary strand. The newly formed oligomer is therefore only accessible in stoichiometric amounts relative to the template, and the complex between the template and the synthetic oligomer needs to be disassembled in a subsequent release step 26 . Catalytic turnover has remained elusive in templated oligomerization. Here we report catalytic oligomerization that uses a synthetic template to bind, activate and covalently link monomeric building blocks in one pot with control over the length of the newly formed oligomer.

Results and discussion
We envisioned the following components and features as key to facilitating a catalytic length-controlled oligomerization (Fig. 1b): (1) a macrocyclic template (T) decorated with two sets of catalytic sites (green and dark blue) located in defined mutual distances on opposite faces of the cavity; (2) a bifunctional monomeric building block (M) bearing two functional groups (blue and light green) that only react with each other upon activation by the catalytic sites of the template; and (3) the formation of an oligomer (O) that has a lower binding affinity than the monomeric building blocks to the template. Variations of the length of the template, and thereby the number of bifunctional catalytic sites, should then activate a different number of monomers and lead to the formation of an oligomer with control over its length.
Based on the above considerations, the macrocyclic template must be rigid and built from modular components that allow for facile Article https://doi.org/10.1038/s44160-022-00228-9 oligomerization with a macrocyclic template (Fig. 2c). This aldol-type reaction is closely related to the process nature uses for the enzymatic synthesis of polyketides 29 . The MAHT serves as a thioester enolate surrogate and the release of CO 2 is a driving force. Bifunctional organocatalysts with thiourea and amine moieties 30 catalyse this reaction and yield β-hydroxy thioesters via C-C bond formation, followed by decarboxylation [31][32][33] . We therefore anticipated that macrocyclic templates with thiourea and amine moieties at opposite sides of the cavity should facilitate the length-controlled oligomerization of monomer 1 that bears both an aldehyde and an MAHT group (Fig. 2a,d). The distance between these two moieties in 1 should suffice to span the distance between the two sides of the macrocyclic template. Further, the product β-hydroxythioester, which lacks a carboxylic acid group and thus a coordinating site, should bind significantly less tightly to the thiourea moieties of the template compared to the substrates, and thus dissociate from the catalyst and allow for turnover in the templated oligomerization reaction (Fig. 1b and Fig. 2c,d).
length variations. Further, the template needs to allow for functionalization with catalytic sites at geometrically well-defined positions. We envisioned that oligoproline-based macrocycles would fulfill all of these requirements (Fig. 2a). Already at a chain length of six residues, oligoprolines form the polyproline II (PPII) helix in which every third residue is located at the same face of the helix at a distance of ~9 Å (Fig.  2b) 27,28 . Thus, the catalytic sites can be aligned on the same side of the helical scaffold to point into the cavity. Further, these peptides can be easily prepared with different lengths by standard solid-phase peptide synthesis (SPPS), are soluble in aqueous and organic solvents and can be derivatized if γ-azidoproline residues are used as building blocks 28 .
Macrocycle formation should be possible by connecting two oligoproline moieties, for example, by acyl hydrazone formation, which is a reaction that occurs under mild conditions and is compatible with many other functional groups.
We installed acyl hydrazine and aromatic aldehyde moieties at the terminal proline residues of H1-H3 and H1'-H3', respectively, as sites for the macrocyclization. For the macrocyclization, the two half-macrocycles were reacted in a 1:1 ratio in methanol containing 1% of trifluoroacetic acid (TFA) at a dilution of 2 mM (Fig. 3b). Notably, the macrocyclic templates T1-T3 formed in yields greater than 80% and did not require chromatographic purification (for liquid chromatography-mass spectrometry and 1 H nuclear magnetic resonance (NMR) spectroscopic analyses, see Supplementary Figs. [3][4][5]. Neither larger macrocycles nor linear or polymeric products were observed. The presence of TFA was important for effective macrocyclization, which suggests that the trifluoroacetate anion coordinates to the thiourea and ammonium moieties and preorganizes the two oligoproline halves for cyclization 34 . Circular dichroism spectroscopic analyses confirmed the expected PPII helicity (minimum at ~208 nm and maximum at ~225 nm; Supplementary Fig. 6) of the oligoproline moieties in templates T1-T3.
MAHTs are prone to unproductive decarboxylation without concomitant C-C bond formation and hydrolysis of the thioester in protic solvents and at elevated temperatures 31,35 . We therefore performed the templated oligomerization reactions with bifunctional monomer 1 (prepared from para-hydroxybenzaldehyde, Supplementary Fig. 7) in a mixture of CH 2 Cl 2 :dimethylsulfoxide (DMSO) (20:1), at −10 °C and at a concentration of 10 mM (Fig. 4a). Under these conditions, mainly oligomers are formed along with a small amount of disulphide 2 that arises from hydrolysis and oxidation of 1.
In the presence of 10 mol% of the macrocyclic templates T1-T3, complete consumption of monomer 1 took place. Organocatalytic reactions between MAHTs and aldehydes typically require catalyst loadings of 20-30% to reach full conversion within a similar time period 32,33 .  Article https://doi.org/10.1038/s44160-022-00228-9 Thus, the reactivity of the templates is comparable to that of conventional organocatalysts. Size exclusion chromatography analysis of the products and comparison of the data with pure, authentic samples of di-, tri-and tetrameric oligomers O1-O3 revealed a remarkable control over the length during the oligomerization process (Fig. 4b): template T1 oligomerized 1 only into dimer O1, template T2 formed predominantly trimer O2 (77%) along with traces of tetramer O3 (O2/O3 25:1) and the largest template T3 produced primarily tetramer O3 (66%) together with a smaller amount of trimer O2 (O3/O2 5:1). The identity of the products, which formed with an enantioselectivity of 20-30% enantiomeric excess ( Supplementary Figs. 8 and 9), was further confirmed by 1 H NMR spectroscopic and mass spectrometric analyses of the reaction mixtures and of the pure products after chromatographic separation ( Fig. 4c and Supplementary Fig. 10). Thus, to the best of our knowledge, T1-T3 represent the first catalysts that allow for length-controlled templated oligomerization. The number of catalytic sites within the template correlates with the length of the oligomer (Fig. 4d). Such a catalytic process is fundamentally different from previous oligomerizations, for example via aldol reactions, which used either controlled polymerization or iterative approaches 22,[36][37][38] . Control experiments that used 'half-macrocycles' bearing only thiourea or amine moieties led to little conversion of monomer 1 and the predominant formation of disulphide 2 (Supplementary Figs. 11e,f and 12e,f). A proline hexamer with primary amine and thiourea groups at Pro 2 and Pro 5 partially converted the starting material to disulphide 2 and traces of dimer O1 (Supplementary Figs. 11g and  12g). Similar results were obtained with a catalyst where the amine and thiourea functionalities were separated by a long flexible linker ( Supplementary Figs. 11h and 12h). Furthermore, Takemoto's catalyst, a powerful catalyst for reactions of MAHTs with electrophiles 39 that bears a tertiary amine and a thiourea group, caused almost exclusive decarboxylation of monomer 1 (Supplementary Figs. 11i and 12i). A derivative of Takemoto's catalyst, with a primary instead of a tertiary amine moiety, polymerized the starting material without any length control (Fig. 4c, bottom). The same uncontrolled oligomerization was observed in the presence of bifunctional cinchona alkaloid catalysts with amine and thiourea moieties ( Supplementary Figs. 11j,k and 12j,k). These results show that the length control observed in the oligomerization of 1 by catalysts T1-T3 arises from the structural rigidity of the macrocyclic template and the spatial alignment of the catalytic functionalities inside the macrocyclic cavity. T1-T3 allow access to oligomers of 1 in a one-pot process, which is more efficient than conventional stepwise synthesis. For example, O2 is produced with 54% yield in a single synthetic step when using catalyst T2, whereas four synthetic steps with a total reaction time of 8 days are necessary to prepare O2 from a protected derivative of 1 with an overall yield of 21% ( Supplementary Fig. 13).
Titration experiments in CD 2 Cl 2 : DMSO-d 6 (20:1), the same conditions as used for the catalysis, revealed a binding constant of K a = 23.0 ± 3.0 M −1 for the interaction between template T1 and substrate 1, and a K a of 8.1 ± 1.3 M −1 for the interaction between the product O1 and T1 (Supplementary Figs. 14 and 15). This approximately threefold lower affinity corroborates that the product binds less tightly than the monomers to the template, thus enabling catalytic turnover.

Conclusions
In conclusion, we have created fully synthetic templates capable of constructing linear oligomers from monomeric building blocks in a catalytic one-pot process. The templates can be programmed to produce oligomers of specific lengths by the choice of their dimensions and the number of catalytic sites installed within their cavities. Controlled product formation is achieved through multiple recognition and activation events that facilitate the catalytic process. Thus, the templates serve a dual role of bringing the monomeric components together and catalysing the covalent bond formation reaction between them. These results provide basic insights into the principles of catalysis and oligomerization, key processes for the evolution of life, and should   Fig. 3 | Synthesis of macrocyclic templates T1-T3. a, SPPS of half-macrocycles H1-H3 and H1'-H3'. b, Macrocylization of H1-H3 and H1'-H3' to templates T1-T3. Boc, tert-butoxycarbonyl; Fmoc, fluorenylmethoxycarbonyl; r.t., room temperature. Article https://doi.org/10.1038/s44160-022-00228-9 inspire future research on the controlled construction of complex molecules from individual components.

General protocols for SPPS and off-resin functionalization
The equivalents reported in the procedures refer to the loading of the resin used for SPPS.

N-terminal acetylation
Et 3 N (30 equiv.) and Ac 2 O (50 equiv.) were added to the amino-functionalized resin suspended in CH 2 Cl 2 , the mixture was agitated for 15 min and then washed with CH 2 Cl 2 (5×).
Cleavage from the solid support and side-chain deprotection of the peptides

Desalting of the peptides
The peptide TFA salt was dissolved in methanol. The solution was filtered through a Stratospheres XP desalting cartridge (preactivated with methanol). The cartridge was washed with methanol (4×). All volatiles of the collected fractions were evaporated under reduced pressure.

Coupling of the acetal-protected benzaldehyde moieties
The purified peptide with amino functionalities at both terminal proline residues (1 equiv.) and 4-(1,3-dioxolan-2-yl)benzoic acid pentafluorophenyl ester (7) (8 equiv.) were dissolved in CH 2 Cl 2 . Diisopropylethylamine (8 equiv.) was added and the mixture was stirred at room temperature overnight. Afterwards, the volatiles were removed under reduced pressure and the crude residue was washed with Et 2 O (3×) to remove excess of 7. The crude product was used in the next step without further purification.

Cleavage of the acetal groups
The peptide (50 µmol) was dissolved in methanol (2 ml) and a 1 N aqueous HCl solution was added (2 ml). The solution was stirred at room temperature for 3 h. Then CH 2 Cl 2 (5 ml) and H 2 O (5 ml) were added. The layers were separated. The aqueous layer was extracted with CH 2 Cl 2 (3 × 5 ml). The combined organic extracts were dried over MgSO 4 , filtered and concentrated. The product was purified by filtration over a plug of silica using CH 2 Cl 2 :MeOH (5:1) as eluent.

General protocol for the macrocyclization reactions
The two half-macrocycles (20 µmol each) were dissolved in methanol (10 ml). TFA (100 µl) was added and the mixture was stirred at room temperature for 4 h. Then, all volatiles were removed under reduced pressure and the crude solid was washed with diethyl ether (2 × 5 ml) and CH 2 Cl 2 (2 × 5 ml). The product was desalted and then used directly in the templated oligomerization reactions. Note, it is important to perform the macrocyclization reaction at a 2 mM concentration of both starting materials. At lower concentration, the reaction proceeds cleanly, but at a considerably lower rate. Increasing the concentration resulted in the formation of side products, for example, larger oligomers.

General protocol for the templated oligomerization reactions
The bifunctional substrate 1 (2.25 mg, 10 µmol) was dissolved in CH 2 Cl 2 :DMSO = 20:1 (0.1 ml). The solution was then added to a solution of the macrocyclic template (1 µmol) in CH 2 Cl 2 :DMSO = 20:1 (0.9 ml) at −10 °C. The reaction was subsequently stirred at −10 °C for 5 days. Then, triethylamine (1.5 µl, 10 µmol) was added and the reaction was allowed to warm to room temperature. The volatiles were removed under reduced pressure, without heating. The residue was redissolved in CH 2 Cl 2 and filtered through a plug of silica. The silica plug was washed with CH 2 Cl 2 :MeOH = 50:1 and the collected filtrates were concentrated.