CHEM 496 RESEARCH DESCRIPTION

Report Author: Edgar Luat

Date Submitted: 22 June 2017
Research Mentor: Md Anisur Rahman
Author’s Affiliation: Tang Polymer Group

INTRODUCTION
Antibiotic resistance has become an increasingly concerning issue in healthcare. The
development of super resistant bacteria has rendered traditional antibiotics, such as
penicillin, ineffective, revealing a need for the development of new antimicrobial
compounds that are both cost-effective and non-toxic to humans [1].

This specific project focuses on derivatives of bile acids, and their modification into
functional monomers and polymers with significant antimicrobial activity. The specific
bile acids used were cholic acid, chenodeoxycholic acid, deoxycholic acid, and
lithocholic acid, with similar backbone structures, varying in hydroxyl group number and
placement. These bile acids were first polymerized using RAFT methods, then the
hydroxyl groups were modified by esterification and the attachment of a quaternary
amine. Each intermediate and final compound was verified and characterized using 1H
NMR spectroscopy.

The overall concept is that a modified cationic charge on these amphiphilic bile acids
will allow for the disruption of the cell membranes of bacteria, which carry negative
charges. This experiment aims to measure the relative antimicrobial activity of each of
these derivatives, both as monomer and polymer structures, to create a foundation for
further research into these compounds.

EXPERIMENTAL

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 Figure 1: General reaction scheme using cholic acid

The monomer synthetic and polymerization methods were modelled after the research
conducted by Pal, Roy, and De, which focused on the thermal analysis, solution
behavior, and self-assembly of cholic acid based polymers [2]. The general reaction
scheme for each bile acid derivative is shown in Figure 1 above.

The synthesis of each monomer was prepared using molar ratios of 1:0.1:1.1:1.1 of bile
acid, DMAP, DCC (or EDC), and HEMA respectively. Initially, cholic acid (5 g) and
DMAP were placed into a 100mL round bottom flask with a magnetic stir bar. The flask
was purged under N2 atmosphere, and dissolved in 40mL dry THF. Once fully
dissolved, DCC was added to the flask and dissolved. The system was placed in an ice
bath, and HEMA was added dropwise to the mixture. The ice bath was removed after
30 min, and the reaction was allowed to progress for 48-72 hrs. The THF was
evaporated using a rotary evaporator, the product was dissolved in 40mL DCM, and
extracted using a separatory funnel with two washes of 5% HCl, three washes of
NaHCO3, and two washes of brine solution. The DCM was evaporated, and the sample
was purified using column chromatography with a hexane-ethyl acetate mixture as the
mobile phase. The ratio of hexane and ethyl acetate in the mobile phase was varied
based on the polarity of each respective bile acid, choosing the most effective ratio to

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separate the target compound from HEMA. Once purified, the monomers were
polymerized using RAFT methods, and cationically modified according to the reaction
scheme above. The same method was used for all four bile acid derivatives, and
characterization of the compounds was performed using 1H NMR.

It should be noted that the catalyst, DCC, was also replaced with EDC for several of the
reactions, with varying results for each acid. Ideally, the use of EDC would allow for the
easier purification of the side products from the monomer structures. Furthermore, it
should also be noted that chenodeoxycholic acid formed a viscous gel after each
synthetic step, making the polymerization process difficult to proceed with. Lithocholic
acid showed similar purification difficulties in the column, with a polarity close to that of
HEMA. More experiments should be conducted in order to determine the optimal
conditions for the purification of these compounds, and decide whether these
derivatives are suitable for the synthetic application.

Through my experience working in the laboratory, I have gained both familiarity and
conceptual knowledge of techniques such as liquid-liquid extraction and column
chromatography. I have also seen first-hand how organic compounds are synthesized in
a laboratory setting, respectively applying the mechanistic knowledge previously gained
in Organic Chemistry courses, and have been introduced to the RAFT polymerization
mechanism of synthesizing polymers. Successful monomer synthesis, separation, and
purification was achieved for all four bile acids, and were further polymerized by Anis.
Practical experience was also gained through my continued use of equipment such as
the rotary evaporator, NMR apparatus, and high vacuum pump. Lastly, I have been
introduced to the industry of polymer chemistry, and the promising prospects that this
type of research holds.

FUTURE STUDY
The current project is still in its preliminary stages, and the modified monomer and
polymer structures of all four derivatives must be prepared for adequate comparison of
antimicrobial activity. Once this is completed, further research can be conducted into the
arrangement of these polymers into varying three-dimensional structures such as a
polymer brush, or spherical polymer. Due to the increased surface area of these
polymers, these arrangements often show increased antimicrobial activity in comparison
to the free polymer, where the modified cationic charges are more likely to adhere to the
bacterial cell membrane.

Further study can also be conducted utilizing other terpenoid-based natural biomass
such as glycyrrhetinic acid, derived from licorice plants. This acid on its own has
antiviral and antimicrobial activity, and with cationic modification and polymerization,
could show even further promise in antibiotic synthesis.

REFERENCE

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1. Ganewatta, M. S.; Chen, Y. P.; Wang J.; Zhou, J.; Ebalunode, J.; Nagarkatti M.;
Decho, A. W.; Tang, C. Bio-inspired resin acid-derived materials as anti-bacterial
resistance agents with unexpected activities. Chem. Sci., 2014, 5. 2011.

2. Pal, S.; Roy, S. G.; De, P. Synthesis via RAFT polymerization of thermo- and pH-
responsive random copolymers containing cholic acid moieties and their self-
assembly in water. Polym. Chem., 2014, 5, 1275-1284.