New synthetic approaches for metal salphens and development of microfluidic platforms to study their G-quadruplex binding
File(s)
Author(s)
Rakers, Viktoria Lena
Type
Thesis
Abstract
Guanine-rich sequences are primarily found in the promoter region of several proto-oncogenes and in the telomeric region; they have been shown to readily fold into quadruple-stranded structures, termed G-quadruplexes, in vitro. Mounting experimental evidence suggests that these structures are involved in key biological processes like replication, transcription and telomere maintenance. A wide range of high-affinity binders has been presented in the literature, but the development of small molecule binders has been mostly limited to conventional batch synthesis. In addition, screening efforts for new potent binders are mostly restricted to 96-well plate-based techniques with reduced throughput. The research presented in this thesis aimed to overcome both shortcomings by providing new technology and new synthetic approaches, the key findings are the following:
1) A “one-pot” as well as an “in flow” synthesis approach was developed for metal salphen based G4 binders. 1H-NMR spectroscopy proved full conversion of a range of Ni(II) salphen complexes. The FRET melting data of the “one pot” prepared compounds (which do not require any purification before testing) were in good accordance with the conventionally synthesised compounds.
2) A microfluidics-based FRET melting platform that enables the real-time and on-line assessment of G-quadruplex binders in continuous flow was developed. The performance of the platform was tested with four commercially available G4 binders BRACO19, TMPyP4, PDS and PhenDC3, the non-binder TMPyP2 as negative control and three new Ni(II) salphen complexes.
3) Segmented flow microfluidics was exploited to develop a pressure-driven approach for generating monodisperse water-in-oil droplets which relied on integrating a quartz pipette (pore size = 3 – 10 µm) into a microfluidic device and pushing the dispersed phase at a constant flow rate through the pipette while varying the carrier phase flow rate. Droplet volumes ranged from 231 fL to 261 pL for a single device when varying the carrier phase flow rate from 1 to 500 µl/min. Using this droplet generation method high-throughput fluorescence-based assays in droplets, namely the FRET melting assay and the Job’s method were carried out, to shine light on the stabilising effect of several G4 binders (PDS, BRACO19, TMPyP4) and on the binding stoichiometry of a novel Pt(II) salphen-based “switch-on” ligand.
4) Furthermore, the integration of the biological target (dimeric quadruplex DNA) into the in situ synthesis was explored in a target-guided synthesis approach. Four different alkyne modified Ni(II) salphen complexes and three different diazide PEG linkers served as building blocks for the in situ “click” reaction. Successful protocols to analyse the mixtures by LC-MS were developed. However, integration of the target inhibited the reaction rendering the “target-guided synthesis” approach unsuccessful.
1) A “one-pot” as well as an “in flow” synthesis approach was developed for metal salphen based G4 binders. 1H-NMR spectroscopy proved full conversion of a range of Ni(II) salphen complexes. The FRET melting data of the “one pot” prepared compounds (which do not require any purification before testing) were in good accordance with the conventionally synthesised compounds.
2) A microfluidics-based FRET melting platform that enables the real-time and on-line assessment of G-quadruplex binders in continuous flow was developed. The performance of the platform was tested with four commercially available G4 binders BRACO19, TMPyP4, PDS and PhenDC3, the non-binder TMPyP2 as negative control and three new Ni(II) salphen complexes.
3) Segmented flow microfluidics was exploited to develop a pressure-driven approach for generating monodisperse water-in-oil droplets which relied on integrating a quartz pipette (pore size = 3 – 10 µm) into a microfluidic device and pushing the dispersed phase at a constant flow rate through the pipette while varying the carrier phase flow rate. Droplet volumes ranged from 231 fL to 261 pL for a single device when varying the carrier phase flow rate from 1 to 500 µl/min. Using this droplet generation method high-throughput fluorescence-based assays in droplets, namely the FRET melting assay and the Job’s method were carried out, to shine light on the stabilising effect of several G4 binders (PDS, BRACO19, TMPyP4) and on the binding stoichiometry of a novel Pt(II) salphen-based “switch-on” ligand.
4) Furthermore, the integration of the biological target (dimeric quadruplex DNA) into the in situ synthesis was explored in a target-guided synthesis approach. Four different alkyne modified Ni(II) salphen complexes and three different diazide PEG linkers served as building blocks for the in situ “click” reaction. Successful protocols to analyse the mixtures by LC-MS were developed. However, integration of the target inhibited the reaction rendering the “target-guided synthesis” approach unsuccessful.
Version
Open Access
Date Issued
2018-06
Date Awarded
2018-11
Advisor
Vilar, Ramon
Edel, Joshua
Sponsor
Imperial College London
Publisher Department
Chemistry
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)