Recognition-Encoded Synthetic Information Molecules.

ConspectusNucleic acids represent a unique class of highly programmable molecules, where the sequence of monomer units incorporated into the polymer chain can be read through duplex formation with a complementary oligomer. It should be possible to encode information in synthetic oligomers as a sequence of different monomer units in the same way that the four different bases program information into DNA and RNA. In this Account, we describe our efforts to develop synthetic duplex-forming oligomers composed of sequences of two complementary recognition units that can base-pair in organic solvents through formation of a single H-bond, and we outline some general guidelines for the design of new sequence-selective recognition systems.The design strategy has focused on three interchangeable modules that control recognition, synthesis, and backbone geometry. For a single H-bond to be effective as a base-pairing interaction, very polar recognition units, such as phosphine oxide and phenol, are required. Reliable base-pairing in organic solvents requires a nonpolar backbone, so that the only polar functional groups present are the donor and acceptor sites on the two recognition units. This criterion limits the range of functional groups that can be produced in the synthesis of oligomers. In addition, the chemistry used for polymerization should be orthogonal to the recognition units. Several compatible high yielding coupling chemistries that are suitable for the synthesis of recognition-encoded polymers are explored. Finally, the conformational properties of the backbone module play an important role in determining the supramolecular assembly pathways that are accessible to mixed sequence oligomers.Almost all complementary homo-oligomers will form duplexes provided the product of the association constant for formation of a base-pair and the effective molarity for the intramolecular base-pairing interactions that zip up the duplex is significantly greater than one. For these systems, the structure of the backbone does not play a major role, and the effective molarities for duplex formation tend to fall in the range 10-100 mM for both rigid and flexible backbones. For mixed sequences, intramolecular H-bonding interactions lead to folding. The competition between folding and duplex formation depends critically on the conformational properties of the backbone, and high-fidelity sequence-selective duplex formation is only observed for backbones that are sufficiently rigid to prevent short-range folding between bases that are close in sequence. The final section of the Account highlights the prospects for functional properties, other than duplex formation, that might be encoded with sequence.

H-Bonded Duplexes based on a Phenylacetylene Backbone.

Complementary phenylacetylene oligomers equipped with phenol and phosphine oxide recognition sites form stable multiply H-bonded duplexes in toluene solution. Oligomers were prepared by Sonogashira coupling of diiodobenzene and bis-acetylene building blocks in the presence of monoacetylene chain terminators. The product mixtures were separated by reverse phase preparative high-pressure liquid chromatography to give a series of pure oligomers up to seven recognition units in length. Duplex formation between length complementary homo-oligomers was demonstrated by 31P NMR denaturation experiments using dimethyl sulfoxide as a competing H-bond acceptor. The denaturation experiments were used to determine the association constants for duplex formation, which increase by nearly 2 orders of magnitude for every phenol-phosphine oxide base-pair added. These experiments show that the phenylacetylene backbone supports formation of extended duplexes with multiple cooperative intermolecular H-bonding interactions, and together with previous studies on the mixed sequence phenylacetylene 2-mer, suggest that this supramolecular architecture is a promising candidate for the development of synthetic information molecules that parallel the properties of nucleic acids.

Backbone conformation affects duplex initiation and duplex propagation in hybridisation of synthetic H-bonding oligomers.

Synthetic oligomers equipped with complementary H-bond donor and acceptor side chains form multiply H-bonded duplexes in organic solvents. Comparison of the duplex forming properties of four families of oligomers with different backbones shows that formation of an extended duplex with three or four inter-strand H-bonds is more challenging than formation of complexes that make only two H-bonds. The stabilities of 1 : 1 complexes formed between length complementary homo-oligomers equipped with either phosphine oxide or phenol recognition modules were measured in toluene. When the backbone is very flexible (pentane-1,5-diyl thioether), the stability increases uniformly by an order of magnitude for each additional base-pair added to the duplex: the effective molarities for formation of the first intramolecular H-bond (duplex initiation) and subsequent intramolecular H-bonds (duplex propagation) are similar. This flexible system is compared with three more rigid backbones that are isomeric combinations of an aromatic ring and methylene groups. One of the rigid systems behaves in exactly the same way as the flexible backbone, but the other two do not. For these systems, the effective molarity for formation of the first intramolecular H-bond is the same as that found for the other two backbones, but additional H-bonds are not formed between the longer oligomers. The effective molarities are too low for duplex propagation in these systems, because the oligomer backbones cannot adopt conformations compatible with formation of an extended duplex.

Sequence-Selective Formation of Synthetic H-Bonded Duplexes.

Oligomers equipped with a sequence of phenol and pyridine N-oxide groups form duplexes via H-bonding interactions between these recognition units. Reductive amination chemistry was used to synthesize all possible 3-mer sequences: AAA, AAD, ADA, DAA, ADD, DAD, DDA, and DDD. Pairwise interactions between the oligomers were investigated using NMR titration and dilution experiments in toluene. The measured association constants vary by 3 orders of magnitude (102 to 105 M-1). Antiparallel sequence-complementary oligomers generally form more stable complexes than mismatched duplexes. Mismatched duplexes that have an excess of H-bond donors are stabilized by the interaction of two phenol donors with one pyridine N-oxide acceptor. Oligomers that have a H-bond donor and acceptor on the ends of the chain can fold to form intramolecular H-bonds in the free state. The 1,3-folding equilibrium competes with duplex formation and lowers the stability of duplexes involving these sequences. As a result, some of the mismatch duplexes are more stable than some of the sequence-complementary duplexes. However, the most stable mismatch duplexes contain DDD and compete with the most stable sequence-complementary duplex, AAA·DDD, so in mixtures that contain all eight sequences, sequence-complementary duplexes dominate. Even higher fidelity sequence selectivity can be achieved if alternating donor-acceptor sequences are avoided.

H-Bond Self-Assembly: Folding versus Duplex Formation.

Linear oligomers equipped with complementary H-bond donor (D) and acceptor (A) sites can interact via intermolecular H-bonds to form duplexes or fold via intramolecular H-bonds. These competing equilibria have been quantified using NMR titration and dilution experiments for seven systems featuring different recognition sites and backbones. For all seven architectures, duplex formation is observed for homo-sequence 2-mers (AA·DD) where there are no competing folding equilibria. The corresponding hetero-sequence AD 2-mers also form duplexes, but the observed self-association constants are strongly affected by folding equilibria in the monomeric states. When the backbone is flexible (five or more rotatable bonds separating the recognition sites), intramolecular H-bonding is favored, and the folded state is highly populated. For these systems, the stability of the AD·AD duplex is 1-2 orders of magnitude lower than that of the corresponding AA·DD duplex. However, for three architectures which have more rigid backbones (fewer than five rotatable bonds), intramolecular interactions are not observed, and folding does not compete with duplex formation. These systems are promising candidates for the development of longer, mixed-sequence synthetic information molecules that show sequence-selective duplex formation.

Supramolecular template-directed synthesis of triazole oligomers.

Sandwich complexes formed by two zinc porphyrins and a diamine ligand (DABCO) have been used as a supramolecular template to direct the synthesis of triazole oligomers. Monomer units equipped with two polymerizable functional groups, an alkyne and an azide, were attached to the template via ester bonds between a phenol unit on the monomer and benzoic acid units on the porphyrin. Self-assembly of the zinc porphyrins by addition of DABCO led to a supramolecular complex containing four of the monomer units, two on each porphyrin. CuAAC oligomerisation was carried out in the presence of a chain capping agent to prevent intermolecular reactions between the templated products, which carry reactive chain ends. The templated-directed oligomerisation resulted in selective formation of a duplex, which contains two identical chains of triazole oligomers connecting the porphyrin linkers. The effective molarity for the intramolecular CuAAC reactions on the template is 3-9 mM, and because the triazole backbone has a direction, the product duplex was obtained as a 4 : 1 mixture of the parallel and antiparallel isomers. Hydrolysis of the ester bonds connecting the oligomers to the template gave a single product, the phenol 2-mer, in excellent yield. The introduction of a supramolecular element into the template considerably broadens the scope of the covalent template-directed oligomerisation methodology that we previously developed for the replication of sequence information in synthetic oligomers.

Folding and duplex formation in mixed sequence recognition-encoded m-phenylene ethynylene polymers.

Oligomers equipped with complementary recognition units have the potential to encode and express chemical information in the same way as nucleic acids. The supramolecular assembly properties of m-phenylene ethynylene polymers equipped with H-bond donor (D = phenol) and H-bond acceptor (A = phosphine oxide) side chains have been investigated in chloroform solution. Polymerisation of a bifunctional monomer in the presence of a monofunctional chain stopper was used for the one pot synthesis of families of m-phenylene ethynylene polymers with sequences ADnA or DAnD (n = 1-5), which were separated by chromatography. All of the oligomers self-associate due to intermolecular H-bonding interactions, but intramolecular folding of the monomeric single strands can be studied in dilute solution. NMR and fluorescence spectroscopy show that the 3-mers ADA and DAD do not fold, but there are intramolecular H-bonding interactions for all of the longer sequences. Nevertheless, 1 : 1 mixtures of sequence complementary oligomers all form stable duplexes. Duplex stability was quantified using DMSO denaturation experiments, which show that the association constant for duplex formation increases by an order of magnitude for every base-pairing interaction added to the chain, from 103 M-1 for ADA·DAD to 105 M-1 for ADDDA·DAAAD. Intramolecular folding is the major pathway that competes with duplex formation between recognition-encoded oligomers and limits the fidelity of sequence-selective assembly. The experimental approach described here provides a practical strategy for rapid evaluation of suitability for the development of programmable synthetic polymers.

Sequence information transfer using covalent template-directed synthesis.

Template-directed synthesis is the biological method for the assembly of oligomers of defined sequence, providing the molecular basis for replication and the process of evolution. To apply analogous processes to synthetic oligomeric molecules, methods are required for the transfer of sequence information from a template to a daughter strand. We show that covalent template-directed synthesis is a promising approach for the molecular replication of sequence information in synthetic oligomers. Two monomer building blocks were synthesized: a phenol monomer and a benzoic acid monomer, each bearing an alkyne and an azide for oligomerization via copper catalyzed azide alkyne cycloaddition (CuAAC) reactions. Stepwise synthesis was used to prepare oligomers, where information was encoded as the sequence of phenol (P) and benzoic acid (A) units. Ester base-pairing was used to attach monomers to a mixed sequence template, and CuAAC was used to zip up the backbone. Hydrolysis of the ester base-pairs gave back the starting template and the sequence complementary copy. When the AAP trimer was used as the template, the complementary sequence PPA was obtained as the major product, with a small amount of scrambling resulting in PAP as a side-product. This covalent base-pairing strategy represents a general approach that can be implemented in different formats for the replication of sequence information in synthetic oligomers.

Mix and match recognition modules for the formation of H-bonded duplexes.

Oligomeric molecules equipped with complementary H-bond recognition sites form stable duplexes in non-polar solvents. The use of a single H-bond between a good H-bond donor and a good H-bond acceptor as the recognition motif appended to a non-polar backbone leads to an architecture with interchangeable recognition alphabets. The interactions of three different families of H-bond acceptor oligomers (pyridine, pyridine N-oxide or phosphine oxide recognition module) with a family of H-bond donor oligomers (phenol recognition module) are compared. All three donor-acceptor combinations form stable duplexes, where the stability of the 1 : 1 complex increases with increasing numbers of recognition modules. The effective molarity for formation of intramolecular H-bonds that lead to zipping up of the duplex (EM) increases with decreasing flexibility of the recognition modules: 14 mM for the phosphine oxides which are connected to the backbone via a flexible linker; 40 mM for the pyridine N-oxides which have three fewer degrees of torsional freedom, and 80 mM for the pyridines where the geometry of the H-bond is more directional. However, the pyridine-phenol H-bond is an order of magnitude weaker than the other two types of H-bond, so overall the pyridine N-oxides form the most stable duplexes with the highest degree of cooperativity. The results show that it is possible to use different recognition motifs with the same duplex architecture, and this makes it possible to tune overall stabilities of the complexes by varying the components.

Cooperative duplex formation by synthetic H-bonding oligomers.

A series of flexible oligomers equipped with phenol H-bond donors and phosphine oxide H-bond acceptors have been synthesised using reductive amination chemistry. H-bonding interactions between complementary oligomers leads to the formation of double-stranded complexes which were characterised using NMR titrations and thermal denaturation experiments. The stability of the duplex increases by one order of magnitude for every H-bonding group added to the chain. Similarly, the enthalpy change for duplex assembly and the melting temperature for duplex denaturation both increase with increasing chain length. These observations indicate that H-bond formation along the oligomers is cooperative despite the flexible backbone, and the effective molarity for intramolecular H-bond formation (14 mM) is sufficient to propagate the formation of longer duplexes using this approach. The product K EM, which is used to quantify chelate cooperativity is 5, which means that each H-bond is more than 80% populated in the assembled duplex. The modular design of these oligomers represents a general strategy for the design of synthetic information molecules that could potentially encode and replicate chemical information in the same way as nucleic acids.

Mix and match backbones for the formation of H-bonded duplexes.

The formation of well-defined supramolecular assemblies involves competition between intermolecular and intramolecular interactions, which is quantified by effective molarity. Formation of a duplex between two oligomers equipped with recognition sites displayed along a non-interacting backbone requires that once one intermolecular interaction has been formed, all subsequent interactions take place in an intramolecular sense. The efficiency of this process is governed by the geometric complementarity and conformational flexibility of the backbone linking the recognition sites. Here we report a series of phosphine oxide H-bond acceptor AA 2-mers and phenol H-bond donor DD 2-mers, where the two recognition sites are connected by isomeric backbone modules that vary in geometry and flexibility. All AA and DD combinations form stable AA·DD duplexes, where two cooperative H-bonds lead to an increase in stability of an order of magnitude compared with the corresponding A·D complexes that can only form one H-bond. For all six possible backbone combinations, the effective molarity for duplex formation is approximately constant (7-20 mM). Thus strict complementarity and high degrees of preorganisation are not required for efficient supramolecular assembly. Provided there is some flexibility, quite different backbone modules can be used interchangeably to construct stable H-bonded duplexes.