High-Fidelity Sequence-Selective Duplex Formation by Recognition-Encoded Melamine Oligomers

Abstract

Melamine oligomers composed of repeating triazine-piperidine units and equipped with phenol and phosphine oxide side-chains form H-bonded duplexes. The melamine backbone provides sufficient rigidity to prevent intramolecular folding of oligomers up to three recognition units in length, leading to reliable duplex formation between sequence complementary oligomers. NMR spectroscopy and isothermal titration calorimetry (ITC) were used to characterize the self-assembly properties of the oligomers. For length-complementary homo-oligomers, duplex formation in toluene is characterized by an increase in stability of an order of magnitude for every base-pair added to the chain. NMR spectra of dilute solutions of the AD 2-mer show that intramolecular H-bonding between neighboring recognition units on the chain (1,2-folding) does not occur. NMR spectra of dilute solutions of both the AAD and the ADD 3-mer show that 1,3-folding does not take place either. ITC was used to characterize interactions between all pairwise combinations of the six different 3-mer sequences, and the sequence complementary duplexes are approximately an order of magnitude more stable than duplexes with a single base mismatch. High-fidelity duplex formation combined with the synthetic accessibility of the monomer building blocks makes these systems attractive targets for further investigation.

Figure 1

Duplex-forming oligomers. Intramolecular folding lowers duplex stability and reduces sequence-selectivity: (a) 1,2-folding on a polythioether backbone and (b) 1,3-folding on a polyaniline backbone.

Figure 2

A blueprint for duplex-forming molecules. There are three key structural elements: recognition modules for base-pair formation (red and blue), functionality used for the synthesis of oligomers (green), and the backbone module which connects the three components of a monomer. Implementation in melamine oligomers equipped with a phenol·phosphine oxide base-pairing system is illustrated (R are solubilizing groups).

Figure 3

(a) Stepwise substitution of cyanuric chloride to give monomers required for the formation of melamine oligomers. (b) The X-ray crystal structure of 2,4,6-tris(dimethylamino)-1,3,5-triazine (CCDC ref code HMELAM) shows that access to the nitrogen lone pairs is sterically blocked in peralkylated melamines.

Scheme 1. Synthesis of 2 and 4: (a) TIPSCl, imidazole; (b) 1. i-butylamine, 2. NaBH₄; (c) H₂CO, HCl; (d) 1. MsCl, 2. i-butylamine

Scheme 2. Synthesis of H-Bond Donor Monomers 5 and 8: (a) 2, DIPEA; (b) 1-Boc piperazine, DIPEA; (c) piperidine; (d) TFA

Scheme 3. Synthesis of H-Bond Acceptor Monomers 9 and 13: (a) 4, DIPEA; (b) 1-Boc piperazine, DIPEA; (c) piperidine; (d) TFA

Scheme 4. Synthesis of 15: (a) 1. Isobutylamine, 2. NaBH₄; (b) 1. Cyanuric Chloride, DIPEA, 2. Piperidine

Scheme 5. Synthetic Strategy for the Synthesis of a Mixed-Sequence 3-mer, Where R₁, R₂, and R₃ Represent Different Combinations of the D and A Side Chains

Figure 4

(a) Structure of the AAAA·DDDD duplex. The melamine nitrogen atoms are represented as black dots, and R’s are the solubilizing groups. (b) Side and top views of the energy minimum structure of the AAAA·DDDD duplex (MMFFs with chloroform solvation). H-bonds are indicated by black dotted lines. The solubilizing groups and hydrogen atoms are omitted for clarity.

Figure 5

Relationship between the association constants for duplex formation between length-complementary homo-oligomers measured in toluene at 298 K (K) and the number of intermolecular H-bonds formed (N). The line of best fit shown is log K = 1.1N + 1.5.

Figure 6

202 MHz ³¹P NMR spectra in CDCl₃ at 298 K for dilution of the AD 2-mer (0.1–100 mM). The corresponding spectra of the TIPS-protected AD 2-mer and the AA 2-mer are shown for comparison.

Figure 7

³¹P NMR chemical shifts in d₈-toluene plotted as a function of temperature for the AD 2-mer (green), a 1:1 mixture of AA and DD (red), and a 1:1 mixture of A and D (blue). The concentration of AD was 6 mM, and the concentrations of AA, DD, A, and D were 3 mM, so that the concentration of fully bound duplex was the same in all experiments.

Figure 8

(a) Isothermal titration calorimetry data (circles) for titration of DAD into DDD (green), ADA into AAD (red), ADD into AAD (blue), and AAA into DDD (black). The lines represent the best fits to an isotherm that allows for formation of a 1:1 complex as well as dimerization of the two components. (b) Structures of the duplexes formed by self-association of the guest and the host used in the ITC experiments, and the structures of the duplexes formed in the mixture (color coding as in panel a).

Figure 9

Average ³¹P NMR chemical shifts in d₄-1,1,2,2-tetrachloroethane plotted as a function of temperature for 0.1 mM 1:1 mixtures of sequence-complementary 3-mers: AAA·DDD (blue), AAD·DDA (green), and ADA·DAD (red).

Figure 10

Effects of single A → D and D → A mutations (red) on the stabilities of sequence-complementary duplexes: (a) AAA·DDD, (b) ADA·DAD, and (c) DDA·AAD..

Figure 11

(a) Two mismatched duplexes that are expected to make two H-bonds when the sequences are arranged in a linear fashion but actually form three H-bonds based on ITC measurements. (b) Structure of the DAD·AAD duplex showing how the terminal dangling bases (left) can fold back to make a third H-bond (right). The melamine nitrogen atoms are represented as black dots, and the terminal piperidine and solubilizing groups are not shown. (c) Top and side views of the energy minimum structure of the DAD·AAD duplex (MMFFs with chloroform solvation). H-bonds are indicated by black dotted lines. The solubilizing groups and hydrogen atoms are omitted for clarity