Abstract
Recent studies combining in vivo, brain-level neuroimaging and neurostimulation technology have uncovered unique perspectives on brain activity. Limited studies have focused on combining such technologies at the neuronal level. Rhodamine-based dyes like the Janelia Fluor (JF) dye series are helpful in fluorescence neuroimaging with genetically encoded voltage indicators such as the HaloTag protein system. On the other hand, gold-mediated infrared neurostimulation can target specific neuronal structures, especially when these cellsare coated with a thiol monolayer. Considering that thiolated 5-carboxyrhodamine 110 can adsorb gold nanoparticles (GNPs), this thesis aimed to develop a method for adding thiol groups to JF585 – the JF dye which can produce a high fluorescence yield and lifetime. Rhodamine thiolation may be a way to create an interface between fluorescenceneuroimaging with rhodamines and gold-mediated neurostimulation.A retrosynthetic approach was used to propose a multi-step synthesis for a novel library of sulphur-containing 4-carboxy-JF585 dyes. The proposed multi-step synthesis included the de novo synthesis of the protected anthracenone and bromoterephthalate starting materials of4-carboxy-JF585. These extra steps were necessary for the synthesis of 4-carboxy-JF585, ahigh-cost material. All intermediates underwent preliminary proton nuclear magnetic resonance elucidation. Synthesising the tert-butyl protected bromoterephthalate afforded a 59% yield. On the other hand, the silyl O-protected anthracenone proved challenging to synthesise de novo. The first limitation identified was the initial acidified co-oligomerisationof 3-bromophenol with paraformaldehyde. The highest yield afforded for the optimised reaction was 11%. The next two proposed steps in the series afforded a 31% and a 46%yield, respectively. The only attempt at the final permanganate oxidation step required to produce the silyl O-protected anthracenone did not appear successful. The degradation and delays in delivery of the tert-butyldimethylsilyl chloride (TBSCl) reagent required for the silylO-protection step hindered any further testing of the proposed multi-step synthesis of the silyl O-protected anthracenone.
A proof-of-concept study was designed for the final three steps in the proposed multi-step synthesis involving the thiolation of rhodamine dyes while waiting for the TBSCl replacement. This study aimed to produce a novel library of sulphur-containing carboxyrhodamines. A 5% yield of 4-carboxyrhodamine 110 and a 22% yield of 5-carboxyrhodamine 110 were afforded. A novel method for separating these carboxyrhodamines was developed using normal phase gravity-flow column chromatography (NP-GFCC). Only attempts for synthesising the other carboxyrhodamines were made due to the nature of optimising their de novo synthesis.
The future work prescribed for this study mainly encourages finishing the proposed multistep synthesis for the novel library of sulphur-containing 4-carboxy-JF585. The proof-of concept study could also include carboxyrhodamines with similar structures to 4-carboxy-JF585 to better prove the concept of attaching sulphur-containing groups to its dimethylanthracene moiety. Finally, seeing the successful development of a method for isolating carboxyrhodamines using NP-GFCC, it may be worth investigating how to optimise this protocol for other carboxyrhodamines synthesised de novo. The accomplished work on the thesis demonstrates how it is possible to design a low-cost method for the de novo synthesis of carboxyrhodamines for future, application-specific modifications.
| Date of Award | 5 Oct 2023 |
|---|---|
| Original language | English |
| Supervisor | Stephan Levonis (Supervisor), Stephanie Schweiker (Supervisor), Vasan Ganesh (Supervisor) & Kannan Madhuvanthi (Supervisor) |
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