The concepts of self-replication, autocatalysis and crosscatalysis were introduced in Chapter 1. The ability of a replicator to take part in template-mediated formation of itself, and in the formation of another molecule through a crosscatalytic pathway, relies on the presence of recognition elements engineered within its structure. The strength of the recognition mediating the various interactions in a system affects the capacity of a particular template molecule to initiate and participate in auto- and crosscatalytic pathways. Specifically, a single point recognition event in a replicating system drives
the association of an unreacted building block with the replicator template. Therefore, the strength of this association determines the quantity of template that needs to be formed through the slow, bimolecular pathway, before the assembly of individual components in a catalytically-active complex is possible, enabling efficient replication. The template formed through the reaction of the building blocks within the ternary complex possesses recognition motifs identical to those within the building blocks, and hence, the recognition processes exert influence also over the stability of template duplexes, affecting the level of product inhibition in each system.
As noted in the discussion of the minimal model of self-replication in Chapter 1, adjustments in the temperature at which a replicating system is examined can be exploited to modulate the strength of the recognition processes. For example, the inter- action between recognition sites becomes stronger at lower temperatures, resulting in an increase in the proportion of all recognition-mediated complexes in solution. Amongst the recognition-mediated complexes that a decrease in temperature would affect is also the product duplex—in particular, the temperature decrease would increase the stability of this duplex, result in lower catalytic turnover. For this reason, it is imperative to consider simultaneously the benefit of increasing the proportion of catalytically-active complexes in a system and the downside of enhancing product inhibition. Adjusting the concentration of reagents can similarly alter the efficiency of the recognition event—any recognition-mediated processes will not work efficiently at concentrations below the
Kdfor each recognition process (Kd=1/Ka).
Within the Philp laboratory, one particular recognition motif, based on the hydrogen- bonding-mediated association between an amidopyridine and a carboxylic acid has been explored extensively in the design of self-replicating systems. The strength of the association (Ka) in this recognition motif was studied using1H NMR titrations (0 C
and 10 C, CDCl3,Figure 2.1) by varying186 the substituents on the amidopyridine
unit and the position of the carboxylic acid on the aromatic ring: 4-bromophenylacetic acid and 3-bromobenzoic acid. Figure 2.1 demonstrates a number of principles187 derived from the study that can be exploited in the design of recognition-mediated replication networks and their analysis. Namely, the location of the carboxylic acid can alter the strength of the recognition quite significantly—in each case, the four examined aldehydes associated more strongly with the 3-bromobenzoic acid than with the 4- bromophenylacetic acid. The higherKa values determined for the 3-bromobenzoic acid
than for the 4-bromophenylacetic acid are in line with the pKavalues reported188for
these carboxylic acids in the literature (pKabenzoic acid = 4.21 and pKa phenylacetic
acid = 4.31). Similarly, the methyl groups exert a positive inductive effect on the electron density on the amidopyridine ring—the Ka increases with the number of
methyl groups present. The position of a methyl group on the pyridine ring can also alter the basicity—a methyl group in position 6- on the pyridine ring results in a slightly higher association constant with a carboxylic acid than a methyl group in 4- position. Again, this observation correlates with the basicity of methyl-substituted pyridines—the pKavalue of pyridine (pKa=5.25) increases in the presence of even a single methyl
group: the pKa of 2-methylpyridine is 6.06189 and the pKa of 4-methylpyridine is
5.99.189 The presence of two methyl groups further increases the basicity—the pKaof
2,4-dimethylpyridine isca.6.75.190 0 °C 10 °C 0 °C 10 °C 1570 1200 1810 1520 3750 2950 4030 3470 850 620 900 660 1020 680 1770 1620 Ka / M–1 Ka / M–1 Aldehyde R1 R2 H H H Me Me H Me Me N R1 N O H R2 O CO2H Br Br CO2H N R1 N O H R2 O N R1 N O H R2 O Br Br O H O O H O Ka Ka
Figure 2.1 Association constants determined by 1H NMR (499.9 MHz) titration for the associa- tion between various amidopyridine-based aldehydes and 3-bromobenzoic (right) and 4-bromophenylacetic acid (left), examined at 10 mM, 0 C and 10 C, in CDCl3. Data taken from Ref. 186.
The NMR titration experiments confirmed that an increase in temperature reduces the strength of association in all of the examined amidopyridine–carboxylic acid pairs, demonstrating the possibility of altering the proportion of the complexed and free species within a system, and, thus, also the efficiency of processes relying on the for- mation of catalytically-active complexes. Interestingly, the variation in the number of methyl groups on the amidopyridine unit affected not only the strength of its association with the carboxylic acids but also the solubility properties of the final replicating system, incorporating these recognition units. For example, the amidopyridine unit containing no methyl groups exhibited the weakest interaction and also the worst solubility properties, whereas the opposite was true for the 4,6-amidopyridine-based replicators. The replica-
tors examined in this thesis will be based on the well-established recognition between a 6-methylamidopyridine—benzoic acid and 6-methylamidopyridine—phenylacetic acid—pairs of recognition motives that should provide a compromise between sufficient solubility and strength of association, whilst limiting the problem of product inhibition.