"Metabolic chemistry" involving nucleotide phosphates, carbodiimides, and imidazoles

Basically, there are two ways to provide the fuel to drive a chemical self-replicating system. In the first and simplest mode, the energy comes with the building blocks. In eq. 1 either A or B must carry an energy-rich functionality to enable the formation of template C. More elegantly, the fuel is a reagent added to the building blocks as an external component. Such a reagent is the water-soluble carbodiimide EDC (N-3-Dimethylaminopropyl-N’-ethylcarbodiimide) which is widely employed in template directed chemical ligations of oligonucleotides.

To gain a more quantitative understanding into the metabolic part of our systems, we followed the reactions of various nucleotide phosphates with EDC using 1H-NMR and 31P-NMR kinetics. [17] 1H-NMR allowed to monitor the signal of the N-CH3 groups in both EDC as the fuel and EDU as the waste. Under the conditions chosen the spontaneous hydrolysis (EDC → EDU) is a always a slow process, while the addition of nucleotides lead to a remarkable increase of hydrolysis rate. Obviously, nucleotides catalyze the hydrolysis of the carbodiimide and the nature of the latter resides in the nucleophilic nature of the phosphate group. In an activation step, phosphate adds to the carbodiimide leading to an activated phosphate (EDC + P → P*). The latter is the hydrolyzed to regenerate the catalyst and to release the waste product (P* → P + EDU). The rate constants of both steps were determined by dynamic modeling (1.3.1) and nonlinear data fitting using our SimFit program. Table 1 shows the results obtained for simple nucleoside phosphates. Similar experiments (1.3.2) were carried out with an oligonucleotide 3’-phosphate under conditions fully compatible with self-replication experiments.

Table 1   Activation and deactivation rate constants for simple nucleotides from 1H-NMR experiments. Conditions: T = 31.7 °C, 60 mM MgCl2 , 0.1 M MES (pD = 6.1)

Next, the above reactions were followed in the presence of 1-methylimidazole and the data obtained were simulated by dynamic modeling (1.3.1) and fitted using SimFit. It was found that phosphorimidazolides are formed rapidly from nucleotide phosphates and imidazole derivatives in the presence of an in-situ activator. This reaction behaviour is also to be expected for catalipids.