Bridging organic synthesis and RNA biology: total synthesis of sannamycin B and reversible RNA acylation

Lee, Sungjong

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https://hdl.handle.net/2142/130179 Copy

Description

Title

Bridging organic synthesis and RNA biology: total synthesis of sannamycin B and reversible RNA acylation

Author(s)

Lee, Sungjong

Issue Date

2025-07-17

Director of Research (if dissertation) or Advisor (if thesis)

• Han, Hee-Sun
• Sarlah, David

Doctoral Committee Chair(s)

Han, Hee-Sun

Committee Member(s)

• Burke, Martin D
• Mehta, Angad P

Department of Study

Chemistry

Discipline

Chemistry

Degree Granting Institution

University of Illinois Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Keyword(s)

• Aminoglycoside
• total synthesis
• RNA
• RNA acylation
• RNA stability
• hydrogel
• polyacrylamide

Abstract

The first chapter describes the study toward the enantioselective total synthesis of sannamycin B, an aminoglycoside natural product. To induce the desired stereochemistry of 2-deoxyfortamine (2-DOF) core from benzene, which is achiral, copper-catalyzed 1,2-hydroamination mediated by NHC ligand as a source of chirality was utilized. Further olefin-type functionalization encompassing intramolecular epoxide opening and bromonium-mediated rearrangement furnished the protected 2-DOF core in 13 steps with 4.8% overall yield. Glycosyl donor was synthesized in 12 steps from a chiral pool with 12% overall yield. The end-game synthetic steps including condensation of two fragments and removal of protecting groups remain to arrive at the target molecule. The second chapter discusses the endeavor to employ RNA acylation for enhancing the RNA stability against extreme pH, heat, and nuclease activity that have been the biggest barrier to enable multimodal imaging-based spatial profiling of organ and tissues. RNA was sequentially added and incubated with NAI-N3, an acylating reagent, to “cloak” 14-18 out of 18 free 2’-OH groups which have been reported to spur the RNA degradation. The cloaked RNA was treated with phosphine to reverse acylation via Staudinger reduction and intramolecular lactam formation, thereby “uncloaking” RNA and restoring its biological properties. Although acyl groups on cloaked RNA were shown not to be stable enough at pH 10 and 70 °C for more than 6 hours, the increased stability at high pH and in the presence of ribonuclease activity highlighted the potential for further application of protecting RNA through reversible acylation. The last chapter details the application of RNA acylation chemistry for reversible RNA capture within polyacrylamide-based hydrogel beads. A multifunctional RNA-capturing monomer was developed, incorporating three functional groups: acyl imidazole motietyfor RNA 2’-OH acylation, an acryloyl group for co-polymerization with acrylamide, and an azido group to enable RNA recovery. A 996 nucleotide eGFP-mRNA was conjugated with this RNA-capturing monomer and was encapsulated into 20 micrometer-size droplets using a microfluidic droplet maker and syringe pumps. The droplets were subsequently polymerized to hydrogel beads, followed by thorough washing to remove unbound RNA from hydrogels. The quantities of both captured and unbound RNA were measured using droplet digital PCR (ddPCR)-based quantification method to assess the capture efficiency of the monomer. Unfortunately, however, the percentage of RNA retained in hydrogel beads showed no considerable difference between samples with or without the reversible acylation chemistry. Moreover, the reproducibility of the results was limited due to occasional polymerization failures. Ongoing efforts are focused on optimizing each step of the protocol and ensuring consistent sample preparation and measurement.

Graduation Semester

2025-08

Type of Resource

Thesis

Handle URL

https://hdl.handle.net/2142/130179

Copyright and License Information

Copyright 2025 Sungjong Lee

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