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Publication Title | Lab on a Chip A microfluidic platform to synthesise a G-quadruplex binding ligand

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PAPER www.rsc.org/loc | Lab on a Chip A microfluidic platform to synthesise a G-quadruplex binding ligand†

N. M. Smith,a B. Corry,b K. Swaminathan Iyer,a M. Norreta and C. L. Raston*a

Received 12th February 2009, Accepted 26th March 2009

First published as an Advance Article on the web 20th April 2009 DOI: 10.1039/b902986a

An aromatic triarylpyridine chromophore promotes p-stacking interactions with the terminal G-tetrad in quadruplex DNA, stabilizing the structure and presenting a pathway towards cancer treatment by inhibition of telomerase. An interesting parent compound in this class is the dimethylamino functionalised 40-aryl-2,6-bis(4-aminophenyl)pyridine. However, access to this compound using traditional batch synthetic methodology is limited, due to thermodynamic and kinetic constraints. A novel approach to the synthesis of this compound has been developed, involving dynamic thin films, overcoming a series of competing reactions, effectively controlling chemical reactivity and selectivity.

Introduction

Anticancer agents that target DNA are some of the most effec- tive agents in clinical use and have been reported to significantly increase the survival of cancer patients when used in combination with drugs that have different mechanisms of action.1 Conse- quently, there has been much effort in identifying cancer-specific molecular targets as new generation therapeutics. Guanine-rich sequences that can potentially form quadruplexes occur in the promoter region of certain oncogenes and at the 30-terminus of telomeric DNA, hence making the G-quadruplex a potentially attractive target for selective anti-cancer therapy and drug development.2–10 G-quadruplex structures can be stabilized by specific ligands in a new approach to cancer treatment, aimed at inhibition of telomerase, an enzyme involved in telomere main- tenance and cell immortality.11–18 Triarylpyridines are a class of G-quadruplex binding ligands, which do not bind to duplex DNA.19 The aromatic chromophore promotes p-stacking interactions with the terminal G-tetrad thereby stabilizing the quadruplex structure. The dimethylamino functionalised 40-aryl- 2,6-bis(4-aminophenyl)pyridine is an important parent com- pound to access this class of compounds, however, the direct synthesis of this target is not possible using traditional batch methodology due to a series of competing reactions. Here we show a synthesis route to access this compound involving dynamic thin films, overcoming a series of competing reactions. Furthermore, we demonstrate the high G-quadruplex binding and stabilizing properties of the parent dimethylamino func- tionalised 40-aryl-2,6-bis(4-aminophenyl)pyridine using molec- ular dynamics simulation and the G-quadruplex stabilising property of its derivative using Fluorescence Resonance Energy Transfer (FRET).

aCentre for Strategic Nano-fabrication, School of Biomedical, Biomolecular and Chemical Sciences, Crawley, WA-6009, Australia. E-mail: colin.raston@uwa.edu.au

bSchool of Biomedical, Biomolecular and Chemical Sciences, The University of Western Australia, Crawley, WA-6009, Australia

† Electronic supplementary information (ESI) available: SI1: Materials, SI2: Experimental, SI3: Additional Results and SI4: Supplementary Notes. See DOI: 10.1039/b902986a

Experimental

Materials and methods

All chemicals (p-aminoacetophenone, p-dimethylamino-benzal- dehyde, NaOH, propan-1-ol, PEG300) were purchased from Aldrich and were used without further purification. A Protensive 100 series spinning disc processor (SDP) (detailed below) was used with integrated feed pumps to direct the reactants onto the rotating disc. The solutions were delivered onto the disc surface using feed jets integrated into continuous flow gear pumps (MicroPumps). A grooved steel disc with 100 mm diameter was used, which was manufactured from 316 stainless steel with the grooved disc having 80 concentric engineered grooves equally spaced at a depth of 0.6 mm. The disc rotation was varied from 300–2500 rpm. The samples were collected from the outlet for analysis. 1H NMR (500 MHz) and 13C NMR (125 MHz) spectra were recorded on a Bruker AV500 instrument in 5 mm NMR tubes. Samples were recorded in DMSO-d6 solution in ppm (d) and referenced to the internal residual partially-deuterated DMSO septet at 2.50 ppm (1H NMR) and 39.52 ppm (13C NMR). Compound 4 was recorded in CDCl3 solution in ppm (d) and referenced to the internal residual partially-deuter- ated CDCl3 singlet at 7.26 ppm (1H NMR) and 77.16 ppm (13C NMR). Compound 8 was recorded in MeOD solution in ppm (d) and referenced to the internal residual partially-deuterated multiplet at 3.31 ppm (1H NMR) and 49.00 ppm (13C NMR).

Detailed experimental procedures can be obtained from the ESI section.†

Results and discussion

Molecular dynamics simulation

Amine substituents of triarylpyridine provide active sites for hydrogen bonding and cation dipole interactions with the sugar- phosphate backbone and the loops of the G-quadruplex, with replacement of amine by oxygen centred moieties resulting in a detectable loss of binding and stabilising properties.19 We envisioned that an amino functionalised 2,4,6-triarylpyridine with a dimethylamino group, with multiple polarising and protonation sites, would provide enhanced binding and

This journal is a The Royal Society of Chemistry 2009

Lab Chip, 2009, 9, 2021–2025 | 2021

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