Microfluidic integration of photonic crystal fibers for online photochemical reaction analysis

Liquid-filled hollow-core photonic crystal fibers (HC-PCFs) are perfect optofluidic channels, uniquely providing low-loss optical guidance in a liquid medium. As a result, the overlap of the dissolved specimen and the intense light field in the micronsized core is increased manyfold compared to conventional bioanalytical techniques, facilitating highly-efficient photoactivation processes. Here we introduce a novel integrated analytical technology for photochemistry by microfluidic coupling of a HC-PCF nanoflow reactor to supplementary detection devices. Applying a continuous flow through the fiber, we deliver photochemical reaction products to a mass spectrometer in an online and hence rapid fashion, which is highly advantageous over conventional cuvette-based approaches

Design of photoactivatable metallodrugs : selective and rapid light-induced ligand dissociation from half-sandwich [Ru([9]aneS3)(N–N′)(py)]2+ complexes

The synthesis of the inert Ru(II) half-sandwich coordination compounds, [Ru([9]aneS3)(bpy)(py)][PF6]2 (1, [9]aneS3 = 1,4,7-trithiacyclononane, bpy = 2,2′-bipyridine, py = pyridine), [Ru([9]aneS3)(en)(py)][PF6]2 (2, en = 1,2-diaminoethane), and [Ru([9]aneN3)(en)(dmso-S)][PF6]2 (3, [9]aneN3 = 1,4,7-triazacyclononane), is reported along with the X-ray crystal structure of 1. We investigated whether these complexes have photochemical properties which might make them suitable for use as pro-drugs in photochemotherapy. Complexes 1 and 2 underwent rapid (minutes) aquation with dissociation of the pyridine ligand in aqueous solution when irradiated with blue light (λ = 420 or 467 nm). The photodecomposition of 3 was much slower. All complexes readily formed adducts with 9-ethylguanine (9-EtG) when this model nucleobase was present in the photolysis solution. Similarly, complex 1 formed adducts with the tripeptide glutathione (GSH), but only when photoactivated. HPLC and MS studies of 1 showed that irradiation promoted rapid formation of 1:1 (major) and 1:2 (minor) adducts of the oligonucleotide d(ATACATGCTACATA) with the fragment {Ru([9]aneS3)(bpy)}2+. Density functional theory (DFT) calculations and time-dependent DFT reproduced the major features of the absorption spectra and suggested that the lowest-lying triplet state with 3MLCT character, which is readily accessible via intersystem crossing, might be responsible for the observed dissociative behavior of the excited states. These complexes are promising for further study as potential photochemotherapeutic agents

Application of analytical techniques for the study of metal-based anticancer complexes

Transition metal coordination complexes show great promise as novel therapeutic agents with new mechanisms of action, but their characterisation, and identification of their target sites present significant challenges. In this thesis a variety of new analytical methods is explored for the study of platinum, ruthenium, osmium and iridium anticancer complexes. High performance liquid chromatography (HPLC) was used to determine the relative hydrophobicity of a series of photoactivatable Pt(IV) diazido complexes of the general type trans,trans,trans-[Pt(N3)2(OH)2(R)(F11]. Interestingly the hydrophobicities did not follow trends based on literature Log P values of individual ligands and did not correlate with the cellular uptake or antiproliferative activity of the drugs. Other factors such as the quantum yield of the complex, and the type of DNA adducts appear to be more important for their efficacy. Chromatography and high-resolution mass spectrometry were used to study the formation of platinum adducts on DNA when the most active complex trans,trans,trans-[Pt(N3)2(OH)2(PYridine)2], 8 was irradiated in the presence of short single strand oligonucleotides 14 bases in length. Complex 8 was found to bind to the oligonucleotides as a {Pt(pyridine)2}2+ adduct. Modifying the wavelength of activation from UVA to 420 nm had no effect on the type of adduct formed, but the higher energy irradiation achieved maximum levels of DNA platination more quickly. Changing the sequence of the oligonucleotide suggested that the photoactivated form of 8 does not favour the formation of the 1,2-(GpG) bisadduct formed by cisplatin and other clinically approved platinum based drugs, but may form 1,3-(GpNpG) or 1,3-(ApNpG) adducts, as is the case with other trans-platinum complexes. Chiral chromatography using cellulose- and amylose-based stationary phases successfully separated the enantiomers of a series of organometallic 'piano stool' anticancer complexes. This appears to be the first successful separation of facially chiral Ru(II) arene complexes, the enantiomers of which were stable in solution for over 3 h. In contrast, separated cyclopentadienyl WI) complexes with chiral metal centres epimerized within 2 h in solution at ambient temperature. Under similar conditions the enantiomers of the Os(II) arene complex [Os(n6-p-cym)(4-(2-pyridylazo)-N,N-dimethylaniline)Ir remained stable, as did those of the ruthenium-based complex [Ru(9,10- diydrophenanthrene)(en)C1r. It was shown that it is possible to separate the diasteriomers of [Ru(t)6-para-cymene)(iminopyridine)I], that can also be resolved by crystallisationtechniques, and hence, decrease the time required to separate the enantiomers. This work will therefore allow exploration of the biological properties of some of these enantiomers A novel technique for the rapid irradiation and detection of light¬senstive species was developed. Photonic crystal fibers (PCFs) were coupled to a mass spectrometer using HPLC tubing and fittings. This continuous flow method of analysis was validated using the photaquation of cyanocobalamin. The PCF system was compared to the conventional cuvette-based approach. No significant difference in the species detected by MS could be found, but the PCF system had the advantage of requiring 20 times less sample (25 pL), and only 15 min of irradiation compared to 10 h by conventional methods. The new PCF-MS system was then used to study the interaction of the photoactivateable ruthenium-based drug [{(e-indan)RuC1}201-2,3-dPa2+ with a range of small molecules that acted as models for intracellular components, e.g. 5'GMP for DNA. The nucleobase binding properties were consistent with those previously reported with plasmid DNA by Magennis et al: a small amount of binding took place in the dark in view of the aquation of the mondentate leaving groups but this dramatically increased upon photoactivation and loss of the arene ligands. The complex was also found to bind to glutathione (GSH), which is known to detoxify metal-based drugs, an observation possibly explaining its poor anticancer activity

Rapid screening of photoactivatable metallodrugs: photonic crystal fibre microflow reactor coupled to ESI mass spectrometry.

We explore the efficacy of a hyphenated photonic crystal fibre microflow reactor - high-resolution mass spectrometer system as a method for screening the activity of potential new photoactivatable drugs. The use of light to activate drugs is an area of current development as it offers the possibility of reduced side effects due to improved spatial and temporal targeting and novel mechanisms of anticancer activity. The di-nuclear ruthenium complex [{(η6-indan)RuCl}2(μ-2,3-dpp)](PF6)2, previously studied by Magennis et al. (Inorg. Chem., 2007, 46, 5059) is used as a model drug to compare the system to standard irradiation techniques. The photodecomposition pathways using blue light radiation are the same for PCF and conventional cuvette methods. Reactions in the presence of small biomolecules 5'-guanosine monophosphate (5'-GMP), 5'-adenosine monophosphate (5'-AMP), l-cysteine (l-Cys) and glutathione (γ-l-glutamyl-l-cysteinyl-glycine, GSH) were studied. The complex was found to bind to nucleobases in the dark and this binding increased upon irradiation with 488 nm light, forming the adducts [(η6-indan)Ru2(μ-2,3-dpp) + 5'-GMP]2+ and [(η6-indan)Ru + (5'-AMP)]+. These findings are consistent with studies using conventional methods. The dinuclear complex also binds strongly to GSH after irradiation, a possible explanation for its lack of potency in cell line testing. The use of the PCF-MS system dramatically reduced the sample volume required and reduced the irradiation time by four orders of magnitude from 14 hours to 12 seconds. However, the reduced sample volume also results in a reduced MS signal intensity. The dead time of the combined system is 15 min, limited by the intrinsic dead volume of the HR-MS

Photo-isomerisation of alkenyl complexes of platinum(ii) : structural, spectroscopic, kinetic and computational investigations

In this work UVA and blue light have been used to study photo-isomerisation about the C[double bond, length as m-dash]C double bond in complexes of the type [PtCl(–CH[double bond, length as m-dash]CHAr)(tmeda)] [Ar = C6H5, (E)-2a; 4-CH3O–C6H4, (E)-2b; 3-NO2–C6H4, (E)-2c; and 3-CH3O–C6H4, (E)-2d]. The progress of the reaction has been monitored by NMR spectroscopy following irradiation of the NMR sample. The NMR data have been complemented with X-ray diffractometric analysis of compounds (E)-2a–c and (Z)-2a. The kinetic data clearly indicate that a monomolecular mechanism is operating with the energy of the irradiating light influencing the rate of isomerisation but not the equilibrium composition, which is only slightly in favour of the Z isomer. DFT and TD-DFT theoretical investigations have been carried out to elucidate the nature of the main electronic transitions in the UV-Vis region and the mechanism of the photo-isomerisation reaction appears to proceed through a C[double bond, length as m-dash]C bond twist process similar to that involved in purely organic molecules such as stilbene. In the Z isomer, one ortho proton of the phenyl group can come close to platinum (PtHortho distance of 2.632 Å in (Z)-2a). In the case of 2c, the difference in chemical shift between the two ortho protons varies from 3.30 ppm in the Z isomer, where interaction with Pt is possible, to 0.60 ppm in the E isomer, where such interaction cannot take place. The analysis of the DFT orbitals indicates that the most shifted Hortho is that with a greater positive charge, pointing to an H-bond type of interaction