Research

Background

The controlled synthesis of functional organic macromolecules is an important topic in the areas of materials science and chemical biology. After continuous development in the recent decades, polymeric materials in micrometer to nanometer size have shown great potential in targeted delivery, recognition, triggered response and release, biocatalysis and multivalent enhancement. In addition, the emergence of various living polymerization strategy has significantly facilitated the controlled synthesis of functional polymers, making it much easier for effecting chemistry for macromolecules with desired functional moieties. By combining known synthetic strategies and molecular biology mechanisms, rationally designed novel macromolecules can be used as catalytic, antimicrobial, delivery or targeting moieties, or as tools for studying complicated mechanisms in living organisms. Useful as it is, such design and application has become a frontier in materials science and chemical biology. As researchers and higher education providers, we want to design new strategies for the several aims listed below, trying to promote the application of nano and polymeric materials in chemical biology and medicine, at the same time training new generations of scientists with good skill, innovativess and knowledge in this area.


 

Enzyme-mimicking polymeric catalysts

In all the life forms, enzymes of different structures and functions are essential. By catalyzing different biochemical reactions, they keep metabolism running and sustain life. Enzymes are basically proteins formed from the folding of polypeptide chains, and are called metalloenzymes if metal cofactors are incorporated into the scaffolds. Inside enzymes are hydrophobic cavities, which can selectively bind substrates near the active metal centers, effecting catalytic process and forming the corresponding product with high selectivity and activity. As we can see here, this is essentially a metal-organic catalyst “wrapped” by a polymeric ligand, which provides selectivity over substrates and protection on the metal centers, making this combination so powerful that it can perform highly efficient catalysis in the complicated biological environment.

However, enzymatic reactions are “natural” reactions. Although many decades have passed since Wöhler could artificially make urea that thought to be unobtainable without the mysterious “vital force” and ended the hypothesis of vitalism, people are still unable to perform most reactions as efficient as nature does with enzymes. Many important reactions in chemical and pharmaceutical industry are still so “unnatural”, requiring complicated synthetic and purification processes. Despite the large amount of metal-organic and organometallic catalysts reported and used, there are remarkably few examples that can be used in biological environment (aqueous, neutral pH, with the presence of common biomolecules), and even fewer, if existing at all, cases that can be comparable with enzymes in activity and selectivity.

So what if we combine enzyme and synthetic metal catalyst? It can be a good idea but difficulties are many, too. By combining the two we may achieve: 1) metal-organic or organometallic catalysts that work in aqueous environment with high efficiency, which greatly reduces cost and promotes greenization of industrial processes; 2) artificial metalloenzymes that can assist or replace some natural enzymes, in order to “repair” some problematic biochemical processes; 3) artificial “synthetic machines” that work inside cells or tissues to catalyze “unnatural” reactions, which can be very useful as tools in chemical biology and medicinal chemistry. Although the three aims look markedly different from each other, they share the same difficulty in getting high activity, biocompatibility and stability simultaneously. Some researchers have started trying to introduce artificial metal cofactors into natural protein scaffolds and perform directed evolution as a process of structure optimization. This is clearly learning from the mother nature, in hope to utilize the polypeptide scaffold to enhance the existing catalysts with an enzymatic working style.

Intracellular catalysis has the potential to be one of the most important tool in the field of chemical biology. Highly efficient intracellular catalysis can activate an unnatural reaction in a specific location inside cells, affording a functional molecule on demand. For example, if a macromolecular catalyst has selectivity when entering cells, then a anticancer compound may be synthesized only inside cancer cells. Some molecules, small or large, may be synthesized inside cells directly to avoid possible difficulties in introducing them into the cells. Since the concept of intracellular catalysis was announced in 2006 by Streu and Meggers using a ruthenium catalyst as an example, this field has been advancing with considerable difficulties in expanding catalyst pool, concentration limitation, reactivity, biocompatibility and so on. We want to combine the knowledge from chemical biology, organic chemistry and polymer chemistry to seek a solution for the problems in intracellular catalysis. In short, we hope to generate enzyme-mimicking structures combined with artificial metal cofactors. Inspired by nature, such artificial catalysts can bring solubility and biocompatibility from their hydrophilic exterior and a stable, highly efficient catalytic center from their hydrophobic cavities in the interior, which may lead to a system that allows changes in catalytic centers while keeping the artificial skeleton to give a multifunctional catalytic platform. 

 


 

Design, synthesis and application of multi-mechanism antimicrobial polymers

Although the invention of antibiotics in the 20th century had doubled the life expectancy of humans, bacteria infection is still an important problem worldwide. For example, tuberculosis is a common infectious disease with high mortality rate especially in the developing countries, ranking in the top 10 reasons among all the human deaths on earth. Even in developed countries and regions, large-area infection and multi-drug resistant bacteria infection are still causing many deaths in hospitals. The wide use of antibiotics is also causing more and more severe problems from antibiotic resistance, leading to limitations of the use of many antibiotics in clinical trials, further promoting the need for the development of new antibiotics.

As a hot research area in the recent years, antimicrobial polymers hardly generate any drug resistance. Nonetheless, due to their low therapeutic index, there has not been any case in this category approved by the FDA. In contrast to most small molecular antibiotics that use a “target-and-inhibit” mechanism, the bacteria-killing effect from antimicrobial polymers solely rely on membrane disruption by inserting their positively charged hydrophobic chains into the lipid bilayers of bacteria membrane. Because this process is purely phyiscal and the synthesis of membrane involves so many biochemical processes, it is extremely difficult for bacteria to evolve from random mutations to gain resistance to such a killing process. However, since lipid bilayers also exist in mammalian cells, it is also difficult for antimicrobial polymers to gain enough selectivity toward bacterial membranes, leading to relatively low therapeutic indices. Although it is possible to improve therapeutic index by synthesizing and screening different macromolecular structures, this approach is significantly limited by the similarity of mammalian and bacterial cell membranes. 

One additional advantage of antimicrobial polymers is that they can change the permeability of membranes. On the one hand, due to the disruption of membrane by the polymers, permeability increase of bacterial membranes can occur, allowing small molecule antibiotics and macromolecules to enter the bacteria, giving a potential synergistic effect. On the other hand, antimicrobial polymers can enter mammalian cells with ease, killing intracellular bacterial pathogens. This can be extremely important for some persistent disease with intracellular pathogen involved, such as tuberculosis.

Here we aim to introduce new antimicrobial mechanisms for cationic, antimicrobial polymers, in addition to their traditional membrane disruption mechanism, making them a new generation of antimicrobial complex with mixed mechanism of actions. Such new macromolecules should also enter infected mammalian cells to kill intracellular bacteria, leading to more thorough action and reduce the needed treatment time for diseases caused by intracellular bacteria. We would also like to explore the relationship between macromolecular structures and their antimicrobial effects, laying a theoretical foundation for future development of new antimicrobial materials.


 

Design, synthesis and multivalent enhancement of ligands for rCAG expansion

The abnormally long rCAG expansion is the cause of Huntington’s Disease and many types of spinocerebellar ataxia. The expansion is transcribed from the dCTG expansion on different chromosomes (for different disease types) and shows a gain-of-function mechanism that leads to disease phenotypes. The main target of this project is to look for small molecules that can selectively recognize and bind rCAG expansion, which can be thus deactivated to relieve the disease symptoms.

We will take three steps to achieve the goal: (1) by combining some known structures that can associate with DNA/RNA (such as bisamidines and bisguanidines), and known moieties that can recognize A-A mismatch, we can select a few possible structures with the assistance of in silico screening and synthesize these molecules, which can then be evaluated by electrophoretic mobility shift assay (EMSA) or isothermal titration calorimetry (ITC) to see their affinity and selectivity towards the target RNA expansion. (2) Molecules from the above screening can be functionalized and oligomerized to form multivalent ligands, which provide better affinity and selectivity with the target expansion that are inherently MULTIVALENT REPEATS. The multivalent ligands must be able to penetrate cell membrane and enter the cells to perform their functions, which can be realized by conjugation with cell penetration peptides (CPPs) or CPP mimics. (3) By further functionalizing the multivalent ligands, the macromolecular ligands may be granted the capability of degrading or deactivating the bound RNA through the introduction of amino groups, imidazoles or alkylating agents. In each step, the products (small molecules or macromolecules) can be evaluated by EMSA, ITC and model cell experiments to see their efficacy. Drosophila and mice model may also be used to evaluate the best macromolecular candidates from the gradual screening process.

Due to limited manpower and resource, this project is currently suspended.