Periodic Reporting for period 1 - CF (Catalytic foldamers from dynamic combinatorial libraries using high-throughput methods)
Période du rapport: 2021-05-01 au 2023-04-30
The complexity, and specificity of natural enzymes is beyond what has been achieved by chemists in aqueous medium. However, one strategy is to synthesize foldamers, folded oligomers, that are stabilized through intramolecular interactions. The overall structure of foldamers resembles proteins and therefore provides an opportunity to explore the basic principles of how enzymes gain their activity and specificity. Traditionally, covalent-based foldamers require tedious synthetic efforts to fold only into a limited number of confirmations, typically secondary structures (e.g. helices). The host lab had previously discovered a new system to generate foldamers with more complex folds from simple building blocks using dynamic covalent chemistry. The system relies on the building block reacting with itself to form a library of macrocycles. Since the system is dynamic, these macrocycles can exchange and form larger or smaller sized rings. However, (in some instances) a single large macrocycle (foldamer) can emerge from the library and is stabilized by the fold that is dissimilar to any secondary folds known. Therefore, this system provides an opportunity to combine complex folding and catalysis with minimal effort and design. However, due to the “minimal design” of the fold, high-throughput screens for foldamer formation of simple building block sequences bearing catalytic motifs and catalytic activity were required to identify hit active foldamers.
There were two points of importance to this project. The first comes from a fundamental perspective, as uncovering the basic principles connecting a folded molecule to its function (i.e. catalysis) from a bottom-up approach will aid in the synthesis of de novo life. Often times, a fold is carefully engineered into the molecule giving rise to predicted catalytic activity. However, through the use of dynamic combinatorial chemistry in this project, we can discover new folds and new catalytic potential. This new catalytic activity can then be integrated into a larger system that contains other characteristics of life, such as compartmentalization. This added level of complexity takes us one step closer to de novo life. The second point of importance is from a practical perspective. By discovering new folds and hence new functions through a less resource intensive method via systems chemistry, there is the possibility of discovering foldamer-based catalysts that can be used in an industrial setting. However, the development of new technologies was not a primary goal within the time frame of this project.
The overall aim was to develop high-throughput screening methods to quickly identify catalytic foldamers that emerge from dynamic combinatorial libraries. To reach this goal, four objectives have been identified:
1. Synthetically integrate catalytic motifs into building block structure
This action was fully completed. A full suite of building block structures bearing catalytic motifs were synthesized.
2. High-throughput assay development and initial screen of foldamer formation and catalysis
This action was fully completed. The screening for foldamer formation within a DCL were conducted in 96-well plates and libraries that produced (nearly) a single species were analysed further to determine foldamer size. These libraries were screened for catalytic activity with fluorescent-based assays.
3. High-throughput optimization of mixed building block catalytic foldamers
This action was partially completed. Although mixing of building block libraries were conducted in the hopes of improving catalytic activity, it was not conducted through a high-throughput manner nor did the mixed building block foldamers improve catalytic activity.
4. Characterization of catalytic foldamers by NMR spectroscopy and X-ray crystal structures
This action was partially completed. Hit foldamers from action 2 were characterized by NMR spectroscopy but X-ray crystal structures were only partially obtained (with low resolution).
There were two points of importance to this project. The first comes from a fundamental perspective, as uncovering the basic principles connecting a folded molecule to its function (i.e. catalysis) from a bottom-up approach will aid in the synthesis of de novo life. Often times, a fold is carefully engineered into the molecule giving rise to predicted catalytic activity. However, through the use of dynamic combinatorial chemistry in this project, we can discover new folds and new catalytic potential. This new catalytic activity can then be integrated into a larger system that contains other characteristics of life, such as compartmentalization. This added level of complexity takes us one step closer to de novo life. The second point of importance is from a practical perspective. By discovering new folds and hence new functions through a less resource intensive method via systems chemistry, there is the possibility of discovering foldamer-based catalysts that can be used in an industrial setting. However, the development of new technologies was not a primary goal within the time frame of this project.
The overall aim was to develop high-throughput screening methods to quickly identify catalytic foldamers that emerge from dynamic combinatorial libraries. To reach this goal, four objectives have been identified:
1. Synthetically integrate catalytic motifs into building block structure
This action was fully completed. A full suite of building block structures bearing catalytic motifs were synthesized.
2. High-throughput assay development and initial screen of foldamer formation and catalysis
This action was fully completed. The screening for foldamer formation within a DCL were conducted in 96-well plates and libraries that produced (nearly) a single species were analysed further to determine foldamer size. These libraries were screened for catalytic activity with fluorescent-based assays.
3. High-throughput optimization of mixed building block catalytic foldamers
This action was partially completed. Although mixing of building block libraries were conducted in the hopes of improving catalytic activity, it was not conducted through a high-throughput manner nor did the mixed building block foldamers improve catalytic activity.
4. Characterization of catalytic foldamers by NMR spectroscopy and X-ray crystal structures
This action was partially completed. Hit foldamers from action 2 were characterized by NMR spectroscopy but X-ray crystal structures were only partially obtained (with low resolution).
A suite of aqueous-active amine-based nucleophilic catalytic motifs were successfully integrated into the building block structures. These building blocks were successfully synthesized and purified. They were tested for preliminary foldamer formation using chromatography and mass spectroscopy high throughput screens. Many building blocks were tested in a wide range of possible conditions that might influence the outcome of the library. The conclusions from the screen revealed that the full suite of catalytic motifs were tolerated by the system as single large macrocycle libraries were identified. Further characterization of these libraries demonstrated that the large macrocycles identified exhibited foldamer-like properties. The catalytic activity of sequences that proved to form foldamers were tested through fluorescent/colorimetric assays in a high throughput fashion. Assays to screen for different reactions were adapted from literature. From the assays, two classes of foldamers showed activity for two separate reactions. The first class of foldamer showed activity in catalysing retro-Knoevenagel condensations only when the foldamer contained a specific catalytic motif. The origins of this motif-selectivity arose from a small pKa perturbation induced by the fold which only one motif benefited from. A low-resolution X-ray crystal structure of one foldamer from this class was obtained. A lesson from the crystal structures obtained through a collaboration from LMU was macrocycles of the same size but with different residues fold in the same way. The second foldamer class demonstrated ester hydrolysis activity. This time, the fold induced a much larger pKa perturbation. One foldamer from this class was further developed to enhance substrate binding. However, the corresponding product inhibited the foldamer catalysis due to strong binding. Another component was added to the system in hopes to mitigate this problem and also introduce another characteristic of life. This system developed into a unique opportunity to integrate the characteristics of life (compartments and metabolism) induced by foldamers.
Two research publications are currently planned, and the writing of both manuscripts is underway. Once the manuscripts are finalized and published, this will be highlighted on the group’s webpage and Twitter feed. Additionally, the research has already been presented to the scientific community via an oral presentation during an international conference and as a poster presentation in a national conference.
Two research publications are currently planned, and the writing of both manuscripts is underway. Once the manuscripts are finalized and published, this will be highlighted on the group’s webpage and Twitter feed. Additionally, the research has already been presented to the scientific community via an oral presentation during an international conference and as a poster presentation in a national conference.
Understanding the complexity and the origins of life is a fascinating subject for both the general public and scientist alike. This is the first example of foldamers that emerge from dynamic combinatorial libraries with complex folds that induce catalytic function. We have shown that the fold is responsible for the catalytic function via pKa perturbation of the catalytic motif. A general set of rules on building block design to incorporate various catalytic motifs have been established. Additionally, a chromatography based high-throughput screen was developed and can be used in future efforts of finding DCL-derived foldamers. Another key (and unexpected) aspect of this project was the formation of compartments through the catalytic activity of the foldamers. These two characteristics of life (metabolism and compartments) that arise from a self-synthesizing foldamer have not been reported before.