Length and Sequence-Selective Polymer Synthesis Templated by a Combination of Covalent and Noncovalent Base-Pairing Interactions

Abstract

Information can be encoded and stored in sequences of monomer units organized in linear synthetic polymers. Replication of sequence information is of fundamental importance in biology; however, it represents a challenge for synthetic polymer chemistry. A combination of covalent and noncovalent base pairs has been used to achieve high-fidelity templated synthesis of synthetic polymers that encode information as a sequence of different side-chain recognition units. Dialkyne building blocks were attached to the template by using ester base pairs, and diazide building blocks were attached to the template by using H-bond base pairs. Copper-catalyzed azide-alkyne cycloaddition reactions were used to zip up the copy strand on the template, and the resulting duplex was cleaved by hydrolyzing the covalent ester base pairs. By using recognition-encoded melamine oligomers with either three phosphine oxide or three 4-nitrophenol recognition units to form the noncovalent base pairs, exceptionally high affinities of the diazides for the template were achieved, allowing the templated polymerization step to be carried out at low concentrations, which promoted on-template intramolecular reactions relative to competing intermolecular processes. Two different templates, a 7-mer and an 11-mer, were used in the three-step reaction sequence to obtain the sequence-complementary copy strands with minimal amounts of side reaction.

Figure 1

Template-directed oligomer synthesis using a covalent primer and noncovalent base-pairing interactions. A covalent base pair was used to attach the primer to the template by ester chemistry. H-bonding interactions with the phosphine oxide 3-mer gave the pre-ZIP intermediate, and an intramolecular CuAAC reaction resulted in the formation of the duplex. Hydrolysis of the ester base pair in the cleave step released the product and regenerated the template.

Figure 2

Schematic representations of the (a) ligation process in Figure 1 and (b) generalization to a mixed sequence polymer. Dialkynes are covalently bonded to the template via ester base pairs (gray circles). A diol linker (gray) is used to connect two carboxylic acids (pink), so that a carboxylic acid on the template is directly copied as a carboxylic acid on the copy strand. Diazides are noncovalently attached to the template via H-bond base pairs between phenols (blue) and phosphine oxides (red). The ZIP step represents CuAAC polymerization of the two building blocks. The cleave step represents hydrolysis of the diester linkers, regenerating the template and liberating the copy strand.

Figure 3

REMOs form duplexes via noncovalent base-pairing interactions between phenol and phosphine oxide side chains. R is a solubilizing group.

Figure 4

Structures of the dialkynes, diazides, and templates used to realize the templating process shown in Figure 2b.

Scheme 1. Synthesis of Dialkyne 4

(i) Pd(PPh₃)₄, CuI, NEt₃, toluene, 90°C, 16 h, 45%; (ii) KOH, MeOH:THF 3:1, r.t., 16 h, 80%; and (iii) EDC, DMAP, THF, r.t., 16 h, 88%.

Scheme 2. Synthesis of 4-Nitrophenol Dichlorotriazine 7

(i) 3,4-Dihydro-2H-pyran, p-toluenesulfonic acid, DCM, r.t., 20 h; (ii) isobutylamine, DCM, r.t., 16 h; (iii) NaBH₄, MeOH, r.t., 3 h, 75% over 3 steps; and (iv) DIPEA, THF, −78°C, 2 h, 86%.

Scheme 3. Synthesis of 4-Nitrophenol Dichlorotriazine 9

(i) PMB-Cl, K₂CO₃, DMF, r.t., 20 h, 85%; (ii) 2-ethyl-1-hexylamine, MeOH:CH₂Cl₂ 7:3, r.t., 4 h; (iii) NaBH₄, MeOH:CH₂Cl₂ 7:3, r.t., 24 h; and (iv) cyanuric chloride, DIPEA, THF, −78°C, 1 h, 76% over 3 steps.

Scheme 4. Synthesis of Diazides zAAAz and zDDDz

Scheme 5. Synthesis of 7-mer Template pAAA-C-AAAp (22) and 11-mer template pAAA-C-AAA-C-AAAp (24)

(i) Piperidine, DIPEA, THF, 0°C, 2 h; (ii) piperazine, THF, 60°C, 16 h, 95% over 2 steps; (iii) 10, DIPEA, THF, 0°C, 2 h; (iv) 11, THF, 60°C, 16 h, 91% over 2 steps; (v) 4, [Cu(MeCN)₄]PF₆, TBTA, THF, r.t., 16 h, 48%; (vi) LiOH, THF:H₂O:MeOH 5:2:2, r.t., 4 h; (vii) 3, [Cu(MeCN)₄]PF₆, TBTA, THF, r.t., 16h, 32%; and (viii) 14, [Cu(MeCN)₄]PF₆, TBTA, THF, r.t., 16 h.

Figure 5

UV–vis absorption titration of zAAAz into zDDDz (5 μM) in dichloromethane at 298 K. (a) Overlay of the UV/vis absorption spectra. The initial spectrum is shown in black, and the final spectrum in red. (b) Points are the experimental measurements at selected wavelengths, and the lines are the best fit to a 1:1 binding isotherm that allows for the absorption of the guest (K = 4.7 × 10⁶ M^–1).

Figure 6

UPLC traces of the crude reaction mixtures obtained from polymerization using the 7-mer template pAAA-C-AAAp: (a) starting template; (b) attachment of the dialkyne; (c) mixture of the esterified template (10 μM), 14 (40 μM), and 17 (40 μM) before addition of the copper catalyst; (d) product mixture obtained after the CuAAC reaction (40 μM [Cu(CH₃CN)₄]PF₆, 40 μM TBTA, DCM, r.t., 48 h); and (e) product mixture obtained after hydrolysis with LiOH in THF/H₂O. UPLC conditions: C4 column at 40 °C using gradient of 30–100% of THF/formic acid (0.1%) in water/formic acid (0.1%) over 4 min and then 100% THF/formic acid (0.1%) over 2 min. The UV–vis absorbance at 254 nm is plotted.

Figure 7

(a) ESI-MS of the UPLC peak due to the 7-mer duplex product in Figure 6d. Calculated mass (ESI⁺): 1142.8 [M+5H]⁵⁺, 952.9 [M+6H]⁶⁺, 816.9 [M+7H]⁷⁺, 715.0 [M+8H⁺]⁸⁺, 635.8 [M+9H⁺]⁹⁺, 572.3 [M+10H⁺]¹⁰⁺. (b) ESI-MS of the UPLC peak due to the 7-mer copy strand, zDDD-C-DDDz in Figure 6e. Calculated mass (ESI⁺): 939.1 [M+3H]³⁺, 704.6 [M+4H]⁴⁺, and 563.9 [M+5H]⁵⁺.

Figure 8

UPLC traces of the crude reaction mixtures obtained from polymerization using the 11-mer template pAAA-C-AAA-C-AAAp: (a) starting template; (b) attachment of the dialkynes; (c) mixture of the esterified template (5 μM), 14 (90 μM), and 18 (90 μM) before addition of the copper catalyst; (d) product mixture obtained after the CuAAC reaction (40 μM [Cu(CH₃CN)₄]PF₆, 40 μM TBTA, DCM, r.t., 48 h); and (e) product mixture obtained after hydrolysis with LiOH in THF/H₂O. Side products are marked with asterisks in panels (d) and (e). UPLC conditions: C4 column at 40 °C using gradient of 30–100% of THF/formic acid (0.1%) in water/formic acid (0.1%) over 4 min, and then 100% THF/formic acid (0.1%) over 2 min. The UV–vis absorbance at 254 nm is plotted.

Figure 9

(a) ESI-MS of the UPLC peak due to the 11-mer duplex product in Figure 8d. Calculated mass (ESI⁺): 1860.1 [M+5H]⁵⁺, 1550.3 [M+6H]⁶⁺, 1328.9 [M+7H]⁷⁺, 1163.0 [M+8H]⁸⁺, 1033.8 [M+9H]⁹⁺. (b) ESI-MS of the UPLC peak due to the 11-mer copy strand, zDDD-C-DDD-C-DDDz in Figure 8e. Calculated mass (ESI⁺): 1604.9 [M+3H]³⁺, 1204.2 [M+4H]⁴⁺, 963.5 [M+5H]⁵⁺, and 802.9 [M+6H]⁶⁺.

Figure 10

Structures of the two truncated side products observed in polymerization reactions using the 11-mer template.

Figure 11

Displacement equilibria in partially zipped intermediates account for the two side products observed in polymerization reactions using the 11-mer template.

Figure 12

Three-step cycle for sequence-selective synthesis of an 11-mer using a complementary template.