A revised synthesis of the cationic lipids DMRI and MLRI

Author

Benjamin A. Kostic, University of LouisvilleFollow

Date on Senior Honors Thesis

5-2024

Document Type

Senior Honors Thesis

Degree Name

B.S.

Department

Chemistry

Author's Keywords

Lipid nanoparticles (LNPs); Cationic lipids; Gene therapy; MLRI; DMRI

Abstract

Lipid nanoparticles (LNPs) are an increasingly utilized method of intracellular delivery for nucleic acids. The COVID-19 vaccines from Moderna and Pfizer-BioNTech have used LNP technology to combat the recent global SARS-CoV-2 pandemic, saving millions of lives.^1,2 These vaccines consist of a lipid formulation that binds to the anionic phosphate groups of mRNAs and serves as an effective way to transport the nucleic acid across membranes into cells.⁹The mRNA encodes a viral protein. When introduced to cells, the viral protein is produced so that the immune system then can generate an appropriate antibody response.³ However, this approach — genetic immunization, a type of gene therapy — is still developing and needs improvement, with new lipids continually being discovered to better help transport nucleic acids into target cells. One such group of state-of-the-art, ‘third-generation’ lipids are hydroxylated oxime ether lipids (OELs), with emphasis on lipid OEL4.⁴

Although OEL4 formulations have been proven to be an effective transport agent for small interfering RNA (siRNA) in animal model experiments, there are still questions of whether OEL lipids are superior to previous lipid-based delivery systems, such as the efficacious second-generation lipids DMRI (dimyristoyl Rosenthal inhibitor) and MLRI (dissymmetric myristoyl and lauroyl Rosenthal inhibitor), especially in tissue systems not yet well studied.^5,6As a result, our long-range goal is to directly compare synthesized OEL4 to both MLRI and DMRI in order to further analyze their respective abilities to deliver genes to the central nervous system (CNS), an area of study in which ortho ester lipids have not been examined. However, what is of particular interest to the present thesis work is the refinement of a synthetic route to both MLRI and DMRI.

By circumventing multiple steps in the known synthesis of MLRI and DMRI, there is the possibility of developing a better way for synthesizing these molecules that would reduce both time and cost, which is of great interest for obvious reasons. Thus, the immediate goal of this work is to examine a new synthesis of MLRI and DMRI to make these lipids available for the key comparative study with OEL4. In this project, the quaternary ammonium moiety (R₄N⁺ portion of molecule responsible for the characteristic RNA-binding properties of the molecule) of OEL4, MLRI, and DMRI will also be assessed, and their effectiveness will be hypothesized in comparison to newer LNP formulations, such as those using eiOEL4.

The key difference between eiOEL4 and OEL4, MLRI, and DMRI is that eiOEL4 has a tertiary amine core instead of a quaternary ammonium salt. eiOEL4 is also different from OEL4 in that the carbon chain between the oxime ether linkage and the tertiary amine is longer, having been increased from two carbons to five, hence the designation eiOEL4 (extended, ionizable OEL4).

The hypothesis of this thesis is that the protection group strategy (i.e., trityl-group protection of a primary alcohol) used in the literature synthesis of MLRI is unnecessary and can be circumvented. Eliminating this step has the potential to save two linear steps in the synthesis sequence and thereby lower the cost of overall lipid production. The lipids will also be assessed in comparison to the “newer generation” lipids in future studies and whether the newer formulation of this LNP is better than the classical molecules for gene delivery.

We confirmed that the trityl-group strategy employed in the synthesis of MLRI is not needed. Along with this refinement, we examined the synthesis of DMRI by using an excess of acid chloride in the first acylation step, further reducing a step in the synthesis of this lipid. However, due to small scale reactions, and novice level mistakes, some reaction steps lost yield, which can be minimized in future applications.

Recommended Citation

Kostic, Benjamin A., "A revised synthesis of the cationic lipids DMRI and MLRI" (2024). College of Arts & Sciences Senior Honors Theses. Paper 319.
Retrieved from https://ir.library.louisville.edu/honors/319

Lay Summary

The goal of this thesis is to synthesize two molecules (DMRI and MLRI) that will serve as effective cationic lipids for intracellular delivery of nucleic acids to the central nervous system (CNS). The recent COVID-19 vaccines from Moderna and Pfizer-BioNTech have used similar LNP technology to combat the recent global SARS-CoV-2 pandemic, saving millions of lives.^1,2 These vaccines consist of a lipid formulation that binds to the anionic phosphate groups of mRNAs and serves as an effective way to transport the nucleic acid across membranes into cells.⁹ This research will focus on improving the synthesis of the lipid carriers needed for delivering the nucleic acid. The hypothesis is that the number of steps needed to synthesize DMRI and MLRI can be reduced, and as a result, create a more effective method of synthesis.