ß-Turn Induction by a Diastereopure Azepane-Derived Quaternary Amino Acid.

ß-Turns are one of the most common secondary structures found in proteins. In the interest of developing novel ß-turn inducers, a diastereopure azepane-derived quaternary amino acid has been incorporated into a library of simplified tetrapeptide models in order to assess the effect of the azepane position and peptide sequence on the stabilization of ß-turns. The conformational analysis of these peptides by molecular modeling, NMR spectroscopy, and X-ray crystallography showed that this azepane amino acid is an effective ß-turn inducer when incorporated at the i + 1 position. Moreover, the analysis of the supramolecular self-assembly of one of the ß-turn-containing peptide models in the solid state reveals that it forms a supramolecular helical arrangement while maintaining the ß-turn structure. The results here presented provide the basis for the use of this azepane quaternary amino acid as a strong ß-turn inducer in the search for novel peptide-based bioactive molecules, catalysts, and biomaterials.

H-Bond Templated Oligomer Synthesis Using a Covalent Primer.

Template-directed synthesis of nucleic acids in the polymerase chain reaction is based on the use of a primer, which is elongated in the replication process. The attachment of a high affinity primer to the end of a template chain has been implemented for templating the synthesis of triazole oligomers. A covalent ester base-pair was used to attach a primer to a mixed sequence template. The resulting primed template has phenol recognition units on the template, which can form noncovalent base-pairs with phosphine oxide monomers via H-bonding, and an alkyne group on the primer, which can react with the azide group on a phosphine oxide monomer. Competition reactions between azides bearing phosphine oxide and phenol recognition groups were used to demonstrate a substantial template effect, due to H-bonding interactions between the phenols on the template and phosphine oxides on the azide. The largest rate acceleration was observed when a phosphine oxide 2-mer was used, because this compound binds to the template with a higher affinity than compounds that can only make one H-bond. The 31P NMR spectrum of the product duplex shows that the H-bonds responsible for the template effect are present in the product, and this result indicates that the covalent ester base-pairs and noncovalent H-bonded base-pairs developed here are geometrically compatible. Following the templated reaction, it is possible to regenerate the template and liberate the copy strand by hydrolysis of the ester base-pair used to attach the primer, thus completing a formal replication cycle.

Replication of Sequence Information in Synthetic Oligomers.

The 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.

Effect of backbone flexibility on covalent template-directed synthesis of linear oligomers.

Covalent template-directed synthesis can be used to replicate synthetic oligomers, but success depends critically on the conformational properties of the backbone. Here we investigate how the choice of monomer building block affects the flexibility of the backbone and in turn the efficiency of the replication process for a series of different triazole oligomers. Two competing reaction pathways were identified for monomers attached to a template, resulting in the formation of either macrocyclic or linear products. For flexible backbones, macrocycles and linear oligomers are formed at similar rates, but a more rigid backbone gave exclusively the linear product. The experimental results are consistent with ring strain calculations using molecular mechanics: products with low ring strain (20-30 kJ mol-1) formed rapidly, and products with high ring strain (>100 kJ mol-1) were not observed. Template-directed replication of linear oligomers requires monomers that rigid enough to prevent the formation of undesired macrocycles, but not so rigid that the linear templating pathway leading to the duplex is inhibited. Molecular mechanics calculations of ring strain provide a straightforward tool for assessing the flexibility of potential backbones and the viability different monomer designs before embarking on synthesis.

Capping Strategies for Covalent Template-Directed Synthesis of Linear Oligomers Using CuAAC.

Covalent templating provides an attractive solution to the controlled synthesis of linear oligomers because a template oligomer can be used to define the precise length and sequence of the product. If the monomer units are attached to the template using kinetically inert covalent bonds it should be possible to operate at high dilution to favor intramolecular over intermolecular reaction. However, for oligomerization reactions using copper-catalyzed azide alkyne cycloaddition (CuAAC) this is not the case. The rate-limiting step is formation of an activated copper complex, so any alkyne that is activated by copper reacts rapidly with the nearest available azide. As a result, every time a chain end alkyne is activated, rapid intermolecular reaction takes place with a different oligomer leading to the formation of higher order products. It proved possible to block these intermolecular reactions by adding an excess of an azide capping agent that intercepts the chain end of the growing oligomer on the template. By adjusting the concentration of the capping agent to compete effectively with the unwanted intermolecular reactions without interfering with the desired intramolecular reactions, it was possible to obtain quantitative yields of copy strands from covalent template-directed oligomerization reactions. Remarkably, the capping agent could also be used to control the stereochemistry of the duplex formed in the templated oligomerization reaction to give exclusively the antiparallel product.

Molecular replication using covalent base-pairs with traceless linkers.

A unique feature of kinetically inert covalent base-pairing is that the nature of the chemical information that is transferred can be modulated by changing the chemical connectivity between the two bases. Formation of esters between phenols and benzoic acids has been used as a base-pairing strategy for sequence information transfer in template-directed synthesis of linear oligomers, but the copy strand produced by this process has the complementary sequence to the template strand. It is possible to form a base-pair between two benzoic acids by using a hydroquinone linker, which is eliminated when the product duplex is hydrolysed. Using this approach, covalent template-directed synthesis was carried out using a benzoic acid 3-mer template to produce an identical copy. This direct replication process was used in iterative rounds of replication leading to an increase of the population of the copied oligomer.

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.

Experimental and theoretical studies on the rearrangement of 2-oxoazepane α,α-amino acids into 2'-oxopiperidine ß(2,3,3) -amino acids: an example of intramolecular catalysis.

Enantiopure ß-amino acids represent interesting scaffolds for peptidomimetics, foldamers and bioactive compounds. However, the synthesis of highly substituted analogues is still a major challenge. Herein, we describe the spontaneous rearrangement of 4-carboxy-2-oxoazepane α,α-amino acids to lead to 2'-oxopiperidine-containing ß(2,3,3) -amino acids, upon basic or acid hydrolysis of the 2-oxoazepane α,α-amino acid ester. Under acidic conditions, a totally stereoselective synthetic route has been developed. The reordering process involved the spontaneous breakdown of an amide bond, which typically requires strong conditions, and the formation of a new bond leading to the six-membered heterocycle. A quantum mechanical study was carried out to obtain insight into the remarkable ease of this rearrangement, which occurs at room temperature, either in solution or upon storage of the 4-carboxylic acid substituted 2-oxoazepane derivatives. This theoretical study suggests that the rearrangement process occurs through a concerted mechanism, in which the energy of the transition states can be lowered by the participation of a catalytic water molecule. Interestingly, it also suggested a role for the carboxylic acid at position 4 of the 2-oxoazepane ring, which facilitates this rearrangement, participating directly in the intramolecular catalysis.

Divergent, stereoselective access to heterocyclic α,α-quaternary- and ß(2,3,3)-amino acid derivatives from a N-Pmp-protected Orn-derived ß-lactam.

A suitably protected Orn-derived (3S,4S)-ß-lactam was used as common intermediate in the synthesis of conformationally constrained (3S,4S)-2-oxoazepane α,α- and (2S,3S)-2-oxopiperidine-ß(2,3,3)-amino acid derivatives. Compared to alternative procedures using an N-p-methoxybenzyl group at the 2-azetidinone, the incorporation of a p-methoxyphenyl moiety is crucial for the excellent stereochemical outcomes in the preparation of these heterocyclic amino acids. Chemoselective 7- or 6-exo-trig cyclization was achieved through alternative sequences of Pmp-deprotection/Boc-activation, followed by inter- and intramolecular ß-lactam ring opening, respectively.

Replication of synthetic recognition-encoded oligomers by ligation of trimer building blocks.

The development of methods for replication of synthetic information oligomers will underpin the use of directed evolution to search new chemical space. Template-directed replication of triazole oligomers has been achieved using a covalent primer in conjunction with non-covalent binding of complementary building blocks. A phenol primer equipped with an alkyne was first attached to a benzoic recognition unit on a mixed sequence template via selective covalent ester base-pair formation. The remaining phenol recognition units on the template were then used for non-covalent binding of phosphine oxide oligomers equipped with an azide. The efficiency of the templated CuAAC reaction between the primer and phosphine oxide building blocks was investigated as a function of the number of H-bonds formed with the template. Increasing the strength of the non-covalent interaction between the template and the azide lead to a significant acceleration of the templated reaction. For shorter phosphine oxide oligomers intermolecular reactions compete with the templated process, but quantitative templated primer elongation was achieved with a phosphine oxide 3-mer building block that was able to form three H-bonds with the template. NMR spectroscopy and molecular models suggest that the template can fold, but addition of the phosphine oxide 3-mer leads to a complex with three H-bonds between phosphine oxide and phenol groups, aligning the azide and alkyne groups in a favourable geometry for the CuAAC reaction. In the product duplex, 1H and 31P NMR data confirm the presence of the three H-bonded base-pairs, demonstrating that the covalent and non-covalent base-pairs are geometrically compatible. A complete replication cycle was carried out starting from the oligotriazole template by covalent attachment of the primer, followed by template-directed elongation, and hydrolysis of the the ester base-pair in the resulting duplex to regenerate the template and liberate the copy strand. We have previously demonstrated sequence-selective oligomer replication using covalent base-pairing, but the trimer building block approach described here is suitable for replication of sequence information using non-covalent binding of the monomer building blocks to a template.

Azepane quaternary amino acids as effective inducers of 3(10) helix conformations.

A simple method for the synthesis of an azepane quaternary amino acid in enantiopure form is described. Theoretical, NMR, and X-ray studies indicated that this azepane-derived amino acid is an effective stabilizer of 3(10) helical structures in short peptides.

Replication of a synthetic oligomer using chameleon base-pairs.

Salt bridges were used to attach polymerisable amidine monomers to an oligomeric benzoic acid template. CuAAC oligomerisation reactions in the presence of a benzoic acid 3-mer template gave the amidine 3-mer copy as the major product. Cleavage of ester linkers was used to hydrolyse off the amidine recognition units and convert the product into a benzoic acid 3-mer copy of the original template.

Quaternary α,α-2-oxoazepane α-amino acids: synthesis from ornithine-derived ß-lactams and incorporation into model dipeptides.

To explore further the chemistry of amino acid-derived ß-lactams, their conversion to α,α-heterocyclic quaternary amino acid derivatives is investigated. The latter derivatives, containing 2-oxoazepane as the α,α-substituent, are synthesized by a simple Pd-C-catalyzed hydrogenolysis of Orn(Z)-derived 2-azetidinones. The rearrangement from four- to seven-membered lactam ring is driven by the key intramolecular opening of the 1-Boc-ß-lactam, initiated by 7-exotrig ring closure from the NH(2) of the Orn side chain. The synthetic route is applied to the stereoselective preparation of enantiomerically pure 4-amino-3-methyl-2-oxoazepane-4-carboxylate derivatives, for which the structure and configuration is confirmed by X-ray diffraction. Molecular modeling and NMR experiments indicate that these quaternary amino acids are able to drive the adoption of ß-turn secondary structures when incorporated in model dipeptide derivatives.

Controlled mutation in the replication of synthetic oligomers.

Replication of sequence information with mutation is the molecular basis for the evolution of functional biopolymers. Covalent template-directed synthesis has been used to replicate sequence information in synthetic oligomers, and the covalent base-pairs used in these systems provide an opportunity to manipulate the outcome of the information transfer process through the use of traceless linkers. Two new types of covalent base-pair have been used to introduce mutation in the replication of an oligotriazole, where information is encoded as the sequence of benzoic acid and phenol monomer units. When a benzoic acid-benzoic acid base-pairing system was used, a direct copy of a benzoic acid homo-oligomer template was obtained. When a phenol-benzoic acid base-pairing system was used, a reciprocal copy, the phenol homo-oligomer, was obtained. The two base-pairing systems are isosteric, so they can be used interchangeably, allowing direct and reciprocal copying to take place simultaneously on the same template strand. As a result, it was possible to introduce mutations in the replication process by spiking the monomer used for direct copying with the monomer used for reciprocal copying. The mutation rate is determined precisely by the relative proportions of the two monomers. The ability to introduce mutation at a controlled rate is a key step in the development of synthetic systems capable of evolution, which requires replication with variation.

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.

Two-component assembly of recognition-encoded oligomers that form stable H-bonded duplexes.

A new family of recognition-encoded oligomers that form stable duplexes in chloroform have been prepared. Monomer building blocks composed of dialdehydes functionalised with either a trifluoromethylphenol or phosphine oxide H-bond recognition unit were prepared. The dialdehydes were coupled with diamines by imine formation and then reduction to give homo-oligomers between one and three recognition units in length. Duplex formation was characterised by 19F and 1H NMR titration experiments in toluene and in chloroform. For duplexes formed between length complementary H-bond donor and acceptor homo-oligomers, an order of magnitude increase in stability was observed for every base-pair added to the duplex in chloroform. The effective molarity for the intramolecular H-bonds responsible for zipping up the duplex is 30 mM, which results in the fully assembled duplex in all cases. The uniform increase in duplex stability with oligomer length suggests that the backbone structure and geometry is likely to be compatible with the formation of extended duplexes in longer oligomers.

Cap control: cyclic versus linear oligomerisation in covalent template-directed synthesis.

Covalent template-directed synthesis was used to oligomerise monomer building blocks in a controlled manner to give exclusively the linear trimer. Competing reaction pathways were blocked by addition of a large excess of a monomeric capping agent. At a concentration of 1 mM, the cap selectively prevented further reaction of the product chain ends to give polymeric and macrocyclic products, but did not interfere with the templating process.

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.