Abstract
ConspectusThe holy grail identified by Orgel in his 1995 Account was the development of novel chemical systems that evolve using reactions in which replication and information transfer occur together. There has been some success in the adaption of nucleic acids to make artificial analogues and in templating oligomerization reactions to form synthetic homopolymers, but replication of sequence information in synthetic polymers remains a major unsolved problem. In this Account, we describe our efforts in this direction based on a covalent base-pairing strategy to transfer sequence information between a parent template and a daughter copy. Oligotriazoles, which carry information as a sequence of phenol and benzoic acid side chains, have been prepared from bifunctional monomers equipped with an azide and an alkyne. Formation of esters between phenols and benzoic acids is used as the equivalent of nucleic base pairing to covalently attach monomer building blocks to a template oligomer. Sequential protection of the phenol side chains on the template, ester coupling of the benzoic acid side chains, and deprotection and ester coupling of the phenol side chains allow quantitative selective base-pair formation on a mixed sequence template. Copper catalyzed azide alkyne cycloaddition (CuAAC) is then used to oligomerize the monomers on the template. Finally, cleavage of the ester base pairs in the product duplex by hydrolysis releases the copy strand. This covalent template-directed synthesis strategy has been successfully used to copy the information encoded in a trimer template into a sequence-complementary oligomer in high yield.The use of covalent base pairing provides opportunities to manipulate the nature of the information transferred in the replication process. By using traceless linkers to connect the phenol and benzoic acid units, it is possible to carry out direct replication, reciprocal replication, and mutation. These preliminary results are promising, and methods have been developed to eliminate some of the side reactions that compete with the CuAAC process that zips up the duplex. In situ end-capping of the copy strand was found to be an effective general method for blocking intermolecular reactions between product duplexes. By selecting an appropriate concentration of an external capping agent, it is also possible to intercept macrocyclization of the reactive chain ends in the product duplex. The other side reaction observed is miscoupling of monomer units that are not attached to adjacent sites on the template, and optimization is required to eliminate these reactions. We are still some way from an evolvable synthetic polymer, but the chemical approach to molecular replication outlined here has some promise.
Highlights
Sequence information is the basis for the transmission of biological inheritance and the expression and regulation of biological function
In vitro molecular evolution has been harnessed for the development of evolutionary processes to search chemical space for new functional biopolymers[6−8] and to optimize existing biopolymers for therapeutic or manufacturing applications.[9−12] These technologies all rely on nucleic acid replication, because no other system capable of sequence information transfer is currently known.[13,14]
Extending molecular evolution principles to synthetic polymers would allow the exploration of different regions of chemical space and the discovery of new polymer architectures where function is defined by sequence.[15]
Summary
Sequence information is the basis for the transmission of biological inheritance and the expression and regulation of biological function. The irreversibility of the duplex cleavage reaction ensures that there is no possibility of product inhibition in multiple rounds of a replication cycle These kinetically inert covalent base pairs have distinctly different properties from dynamic covalent base pairs, because the chemical steps used for attachment and cleavage are not under equilibrium control, which is an essential requirement if the competing processes highlighted in Figure 2 are to be avoided. By assembling pre-ZIP intermediates where one of the terminal monomers was precapped to remove either the azide or the alkyne functionality (Figure 6), it was possible to directly study CuAAC reactions in which only one of the parallel or antiparallel duplexes can be formed.[2] Titration of 4-tertbutylbenzyl azide into the reaction mixtures was used to determine values of EM for the intramolecular reactions that zip up the duplex, based on the concentration of the external capping agent required to compete with the intramolecular process (Figure 6b). The population of different sequences present in the product mixture obtained after the seven-step replication cycle can be accurately predicted based on statistical incorporation of the mutator monomer
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