| By merging features of combinatorial chemistry and self-assembly processes, dynamic combinatorial chemistry (DCC) offers access to a wide range of substances assembled from relatively small libraries, without the need to synthesize each substance individually. The expression of the products in the dynamic combinatorial library (DCL) is governed by thermodynamics and as a consequence, specific changes in the environmental parameters can lead to the amplification of the "bestfitted" constituents. This selection aspect is usually based on recognition processes according to the Emil Fisher lock-and-key principle. This is of particular interest for drug discovery purposes because the presence of molecular targets such as enzymes can discriminate their own best inhibitor through an in situ dynamic screening of the equilibrating mixture. Over the past ten years, other investigations on DCC by different research groups have highlighted the wide potentialities of the main concept in various fields of chemical research going from self-assembly of inorganic architectures, to receptor generation and substrate binding, as well as catalyst screening. Finally, DCC has also been recently shown to respond to other external chemical or physical stimuli such as protons, phase transition, temperature, or electric field modulations, thus opening interesting potentialities for material science.Self-replication is considered as one of the three prerequisites together with the abilities to metabolize and to undergo mutations that characterize evolving systems. Although nature's property to duplicate macromolecules in cells involves extremely complex machineries, biologists and chemists have been interested in much simpler self-replicating systems in their quest to bridge the gap with a possible origin of life. These minimal self-replicating systems are governed by kinetics and are ideally defined along a three-step model in which a product molecule is self-complementary and directs its replication. In the first step, the product/template reversibly binds its two former components which, due to their spatial proximity in the termolecular complex, react with a minimal loss of entropy in a catalyzed - non-reversible-second step. At the last step, the reversible dissociation of the final complex generates two free molecules of template that initiate two new replication cycles, thus leading to a self-catalytic behavior, i.e. displaying a sigmoid concentration-time profile.In the thesis we have described a new chemical model system which brings together self-replication and dynamic combinatorial reorganization by kinetically and thermodynamically selecting a product - the one capable of duplication - from a pool of reshuffling constituents, according to the simplified process illustrated in Figure 1.A set of building blocks, capable of binding by reversible bonds, produces a dynamic combinatorial library of constituents. Among these products, one can undergo a self- duplicating by templating its two former components. The overall system being kinetically and thermodynamically driven to the formation of the key product, the concentration of the self-replicated product is amplified at the equilibrium by taking back components from the pool of constituents.We use the term "self-replicate" for auto-catalytic systems displaying a sigmoid concentration-time profile and the term "self-duplicate" for a system displaying the general property to thermodynamically or kinetically (or both) favor its own formation. 1. Design of the system that can bring together self-replication/self-duplication and dynamic combinatorial chemistryIn order to design such a system, we envisioned several building blocks capable of i) reversible covalent associations and ii) displaying complementary supramolecular units in order to produce a template with self-recognition properties. The key molecule was chosen as imine Al1Am1, synthesized by the condensation of aldehyde Al1 (derived from a Kemp's imide) and adenosine amine Am1. This self-complementary dynamic compound, inspired by a related "non-dynamic" isosteric system described by Julius Rebek, is able to strongly associate with itself into complex [Al1Am1]2 (Scheme 1). Its dimerization in a chloroform solution through hydrogen bonding was confirmed by 1H-NMR. The presence of homodimer [Al1Am1]2 in solution was also confirmed by mass spectroscopy (ESI-TOF). While Al1 contains a free imide bond on the Kemp recognition group, Al2 is protected by a methyl group on the nitrogen which prevents the formation of hydrogen bonds with the adenine moiety. Al3 is also an analogue of Al1 but without a Kemp's recognition group and with an acetyl group instead, in order to display similar activation energy as Al1 for the condensation reaction with amines. Am1 and Am2 also present close structures, but in the latter, the hydrogen bonds with the Kemp's imide are restricted by the protection of the adenine with a benzyl group.2.Synthesis of all the building blocksBefore investigation of self-replication/self-duplication and dynamic combinatorial chemistry, all the building blocks we had designed were synthesized. The methods for synthesis of all the building blocks(including biphenyl system) were shown in scheme 2 and scheme 3. In addition, all the target products and most of the intermediates were confirmed by 1H NMR, 13C NMR and Exact Mass Spectrometry (ESI-TOF). 3. Investigation on minimal self-replicating of formation of key compoundAl1Am1These comparative analyses with protected and unprotected recognition groups, in various solvents, and together with the characteristic chemical shifts in 1H NMR, indicates that much higher initial rate of formation of Al1Am1 in pure chloroform (5.9 mM.h-1) are supposed to be related to molecular recognition. These amplifications can be the consequences of several mechanistic channels. There were some controversial discussions between Rebek, Menger and Reinhoudt about the initial amide-type self-replicator designed by Rebek in which the amide bond can itself catalyze the reaction. In our case, we do not obtain sigmoid time-dependent profile in pure CDCl3 or acceleration of the reaction in the presence of the product, but finally we got the conclusion that the intramolecular Al1/Am1 condensation is the major channel. Fortunately, the system displays an extremely slight exponential growth in the first 10% of the reaction by decreasing the product inhibition effect in a CDCl3:DMSOd6 (1:0.4) solution (Figure 2). This observation was correlated by the fact that the product Al1Am1 catalyzes the condensation of Al1 and Am1, as is shown in Figure 2, which describes the time course of the reaction upon the addition of initial amounts of product. These results show that the Al1 Am1 replicator is able to catalyze its own formation in a CDCl3:DMSOd6 (1:0.4) solution, although it importantly does not contain an amide bond in our case but an isosteric imine on. Moreover, we can conclude that [Al1Am1]2 need to disassociate to sufficient free Al1Am1 to replicate its formation. 4. Self-Duplicating Amplification in Dynamic Combinatorial LibrariesWe then turned to the thermodynamic study of the DCLs described in Scheme 1.The first library (DCL0) was set up by mixing Al1, Al2and Am1 (15 mM each at 22℃in CDCl3). In this small library, the production of Al1 Am1 (11.68 mM)which formed homodimer [Al1Am1]2 leads to a much stronger bias in the expression of the constituents than Al2Am1 (2.71 mM) at equilibrium. From the this small library, we deduced the depressed energy of homodimer [Al1 Am1] 2 compared with free Al1 Am1 via self-complementary hydrogen bonding, which is 11. 1KJ·mol-1.We mixed Al2, Al3, Am1, and Am2 (15 mM each at 22℃in CDCl3) as the DCL1. in order to determine the distribution of the 8 library members at equilibrium (Figure 3, black bars). The isoenergetic nature of DCL1 is here clearly demonstrated by the statistical distribution of the products (equal expression of the 2 amines and 2 aldehydes (3.4 mM each) and equal expression of the 4 imines Al2-3Am1,2 (5.8 mM each)). This reflects, as expected, the absence of specific supramolecular interactions between the library members.In the experiment (DCL2), Al1, Al2, Al3, Am1 and Am2 were mixed together (15 mM each at 22℃in CDCl3). In this library, the production of homodimer [Al1Am1]2 also leads to a strong bias in the expression of the constituents at equilibrium, far from the statistical distribution (Figure 3, gray bars). In the experiment (DCL3). we divided the process in two steps: i) set up of a pre-equilibrated 8 member library without Al1 (DCL1, Figure 1, black bars), and then ii) addition of Al1 (15 mM) to this library. In DCL3, it takes five times longer to reach equilibrium (22 days) compared to DCL2 (t = 109 h), but the competition produces an identical distribution of constituents in both DCL2 and DCL3 (Figure 3, gray bars for both conditions). These two experiments confirmed the good thermodynamic control of the overall system in these conditions. By comparing the "non self-duplicating" DCL1 (black bars) and the "self-duplicating" DCL3 (gray bars), we can conclude on the following features. First, the expression of the self-duplicator Al1Am1 (9.03 mM) is increased by more than 80% compared to the theoretical statistical distribution. Second, this amplification of the self-duplicator is realized by the takeover of the resources of its direct competitors, i.e. the imines having antagonistic connectivity with (namely Al1Am2, Al2Am1, and Al3Am1, (3.0 mM each). Thus, the amplification of the self-duplicator Al1Am1 compared to its direct competitors reaches a value of +200%.We also studied the kinetic behaviour of the 11 member library DCL2 by plotting the concentration of the constituents as a function of time (Figure 4). In the duplicating DCL2, the self-duplicator Al1Am1 is produced with a V0 of 59×10-1 mM.h-1, which is about 60 times faster than the condensation of Al2Am1, and Al3Am1 (0.96×10-1 mM.h-1); 13 times faster than Al2Am2 and Al3Am2 (4.6×10-1 mM.h-1); and 6 times faster than Al1Am2 (9.6×10-1 mM.h-1). These differential rates lead to a maximum of amplification (kinetic amplification) of the self-duplicator (10.98 mM) much more than other imines at t = 16 h (Figure 3 striped bars). We assume that the kinetic amplification is mainly the result of a pre-association complex between Al1 and Am1 as indicated by the kinetic studies of the individual reactions.In conclusion, the expression of the components in the library evolves along both kinetic and thermodynamic biases that both lead to the amplification of the best duplicator.5. Study of the combinatorial set, in which the naphthalene group was replaced with the biphenyl groupWe set up the library(DCL4) by mixing B1, B2, B3, Am1 and Am2 (Scheme 4) .Compared with scheme 1, the naphthalene group was replaced with the biphenyl group in the molecules of aldehydes(B1, B2, B3). Different from DCL2, the concentrations of the members in DCL4 at each time could not be determined exactly from 1H NMR data, because of the signals were overlapped with each other, although the rate of formation of B1Am1 seemed to be higher than Al1Am1. Fortunately, we could get the rough information from 1H NMR data, so we could conclude that self-duplicating did not make such notable amplification in DCL4 as the combinatorial set described in scheme 1 by reason of less stability of [B1Am1]2.In conclusion, we have demonstrated that it is possible to self-amplify one product in a DCL, namely the one that can self-complementarily direct its own formation. The expression of the components in the library evolves along both kinetic and thermodynamic biases that both lead to the amplification of the best duplicator. Because of the double reversibility of the system (supramolecular H-bonds and molecular imine condensation), the competition is not only ruled by the differential rates of formation of the components, but also by the possible takeover of the building blocks of the antagonistic competitors, thus leading to the decrease of their absolute concentration. From a "Darwinian" point of view, such a system illustrates "molecular evolution" of the most efficient self-duplicator by the destruction of the entities which are not (or less, such as Al1 Am2) able to duplicate themselves.
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