Hydrogen Bond Template-Directed Synthesis of Recognition-Encoded Melamine Oligomers

Abstract

The directed evolution of biopolymers is based on the mutagenic replication of DNA. However, reliance on DNA limits the application of directed evolution to molecules that can be encoded by DNA, such as proteins. The development of a synthetic oligomer which can mutagenically replicate could unlock the power of directed evolution to explore different areas of chemical space. Previous work by the Hunter group has shown that sequence-selective replication of oligotriazoles can be achieved using covalent ester base-pairing chemistry. While successful replication has been achieved with this method, it requires multiple steps, making the replication cycle labour-intensive and low yielding. Here, a new approach was investigated that uses noncovalent H-bonding interactions between 4-nitrophenols and phosphine oxides as the base-pairing chemistry for the template-directed synthesis of recognition-encoded melamine oligomers (REMO). A copper-catalysed alkyne-azide cycloaddition (CuAAC) reaction between two REMO 3-mers, bearing 4-nitrophenol recognition units, an alkyne and an azide, was found to be templated by a complementary REMO 6-mer phosphine oxide template. The effective molarity for the intramolecular reaction that led to the templated product was found to be 2 mM, enabling efficient template-directed synthesis to be carried out at μM concentrations. The major competing reaction was templated macrocyclisation of the linear 6-mer product, which occurred with an effective molarity of 1 mM, limiting the potential of this system for the template-directed synthesis of longer oligomers. The use of a covalently attached primer to cap one end of the copy strand was investigated as a method to intercept the formation of the macrocycle, but macrocycle formation was not completely suppressed. A second approach implemented sequential ligation cycles to avoid competing macrocyclisation. A template equipped with a covalent primer was reacted with complementary REMO 3-mers bearing an azide and a protected alkyne in a H-bond templated CuAAC reaction. Unreacted 3-mers were removed, and the alkyne was deprotected, ready for use in a second ligation reaction. A 10-mer template with a covalent primer was subjected to three ligation cycles and was found to selectively incorporate complementary 3-mers into the copy strand. An advantage of templated reactions that use covalent base-pairing is that they can be carried out at very low concentrations without affecting monomer binding. A new strategy was explored that attached REMO 3-mers to a template by using a combination of H-bond interactions and dynamic covalent imine bonds. Following equilibration, the imines can be reduced, forming a covalent attachment between the template and the 3-mers, which would enable subsequent template-directed synthesis to be carried out at very low concentrations. The chemistry required for a sequence-selective attachment cycle of a REMO 3-mer was successfully demonstrated using this strategy. Finally, a new strategy for the template-directed synthesis of REMO that used active templating of the CuAAC reaction was investigated. A 7-mer rotaxane template was subjected to a templating cycle. Firstly, a macrocycle was covalently attached to the template. Addition of complementary REMO 3-mers and copper catalyst resulted in active templating to form a handcuffed duplex. Cleavage of the duplex returned the rotaxane template and a complementary rotaxane copy strand.