A MICROFLUIDIC APPROACH TO “GREEN” SINGLET OXYGEN MEDIATED OXIDATION Emily Lumley1, Charlotte Wiles1,2, Charlotte Dyer1, Nicole Pamme1, and Ross Boyle1 1
The University of Hull, Department of Chemistry, Cottingham Road, Hull, HU6 7RX, UK 2 Chemtrix BV, Burgemeester Lemmensstraat 358, 6163JT Geleen, The Netherlands
ABSTRACT We report a novel method for conducting “green” singlet oxygen-mediated oxidations within a microfluidic device by immobilising a photosensitiser (prophyrin) on the channel walls. A strategy for immobilising molecules with an isothiocyanate group onto the amine group of a silanised glass surface was optimised using rhodamine B isothiocyanate (RBITC). The immobilisation method was thus employed for the successful continuous flow oxidation of cholesterol by singlet oxygen chemistry using a photosensitive porphyrin bound to the channel surfaces. Comparison of this method demonstrated that porphyrin immobilisation produced significantly greater efficiency than batch experiments, whilst being more reproducible than solution-phase methodology. KEYWORDS Singlet oxygen, Microfluidic, Photosensitiser, Oxidation, Green chemistry INTRODUCTION Singlet oxygen chemistry is an important component of a large number of chemical reactions, including ene, [2+2], and [4+2] reactions as well as heteroatom and heterocyclic photooxidations[1]. These reactions are widely used in the synthesis of precursors for perfume and in drug discovery, among other applications. Microfluidic devices offer many advantages for conducting singlet oxygen chemistry including higher rates of thermal transfer and shorter path lengths for light penetration. Also the small volumes (nL) are perfect for handling hazardous or expensive reagents[2]; attractive features for increasing the safety and reducing costs of singlet oxygen chemistry, respectively. Additionally, immobilisation of a photosensitiser (which converts oxygen gas to singlet oxygen) in a microchannel has the advantage that oxidation products are not contaminated with photosensitiser, as is often observed in batch reactions and conventional microfluidic reactions where the photosensitizer is in free solution. Thus, the work-up becomes more facile since the product is significantly more polar than the starting material, and produces less solvent waste from purification. The significant reduction in waste products is better for the environment as is the reduced reaction time. Here we combine immobilisation of the photosensitiser with microfluidics to investigate the potential of a microchip for performing singlet oxygen chemistry in continuous flow by immobilising the photosensitiser inside the chip. We compare the procedure to a typical batch reaction and to a microfluidic method in which the photosensitiser is in the reaction mixture rather than bound to the channel surfaces. THEORY The treatment of glass with silanising agents allows the addition of a wide variety of functional groups to the exposed surfaces. These functional groups can be further utilised by immobilising additional molecules or biological components onto the treated surface via covalent bonds resulting in a strongly bound layer of a desired material. Also due to the small path length of a microfluidic channel, and the close proximity of a reagent stream to the channel walls, it is possible for the reagent stream in solution to undergo singlet-oxygen mediated oxidations by photoactivating surfacebound molecules within the microfluidic device. Thus a reagent flowing through a microfluidic device, in which a photosensitiser is bound to the glass surface and exposed to a constant light source, would become oxidised in a short time since the photosensitiser would be continually producing singlet oxygen for the reaction, whereas in solution-phase microfluidic reactions the phtotosensitiser is only irradiated for a short time as the reagents flow through the channel. Cholesterol is an example of a species that is affected by singlet oxygen. Cholesterol is oxidised by singlet oxygen into 5α-hydroperoxycholesterol via Type II photoreactions and by electron or hydrogen transfer, ion or free radical species into 7α- and 7β-hydroperoxycholesterol via Type I photoreactions (Figure 1)[3]. Hence, cholesterol was used to demonstrate the production of singlet oxygen within a microfluidic device featuring immobilised photosensitive porphyrin, and for comparison to conventional batch methods and solution-phase microfluidic reactions.
978-0-9798064-3-8/µTAS 2010/$20©2010 CBMS
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Figure 1: Oxidation of cholesterol to the 5α-hydroperoxycholesterol via type II reaction (top) and 7α/7β-hydroperoxycholesterol via type I reaction (bottom) and subsequent reduction to the hydroxycholesterol with NaBH4 for analysis by HPLC.
EXPERIMENTAL The design (figure 2a), fabricated to a depth of 30 μm in glass using photolithography and wet etching procedures, and featured 16 channels of 160 μm width branching from three convergent inlets and meeting at one outlet. Silanisation was performed by allowing a 5% solution of (3-aminopropyl)triethoxysilane (APTES) in dry toluene to react in the channels for 1 hour before sonicating in toluene. A 1 mM solution of the molecule to be immobilised (Rhodamine B isothiocyanate (RBITC) or 5-(4-isothiocyanatophenyl)-10,15,20-tris(4-N-methylpyridiniumyl)porphyrin triiodide (porphyrin)) was prepared in methanol with the addition of triethylamine to make the reagent basic. The solution was pumped through the chip at 3 μL/min for 3 hours, before sonicating the chip in methanol and drying with a flow of nitrogen to yield channels featuring the immobilised compound on the surface. RBITC was used to investigate the success of the immobilisation process itself, while the porphyrin was the photosensitiser used for the oxidation experiments. Three methods of cholesterol oxidation by singlet oxygen were investigated. An off-chip batch reaction was conducted in which 5 mL of a 20 μM solution of porphyrin (capped with propylamine to protect the reactive isothiocyanate group) was mixed with 5 mL of a 10 mM solution of cholesterol in 1:9 methanol:dichloromethane. The reaction mixture was irradiated with Xe light source (using white light over the full visible spectrum), and supplied with oxygen for 1 hour. An on-chip flow experiment was conducted using the same reaction composition, which was pumped through the chip at two different flow rates (5 μL/min and 2.5 μL/min) with a constant supply of oxygen and irradiation with the same light source. Finally an experiment using the channel-immobilised porphyrin was conducted in which a 5 mM solution of cholesterol was pumped through the chip with a supply of oxygen at the flow rates described for the flow reaction above (Figure 2b). The resulting cholesterol mixture in each case was tested using HPLC (C18 column, 40:60 CH3CN:MeOH mobile phase using a UV detector at 205 nm). In order to determine the % conversion of cholesterol to oxidised products, area-under-the-peak analysis was performed. a)
b)
Photosensitiser-immobilised glass chanels
Reagent Inlets
Flow Outlet
Oxygen Oxidised product
1 mm
Light source Figure 2: Experimental set-up; (a) chip design and (b) schematic of the oxidation procedure.
RESULTS AND DISCUSSION The immobilisation of RBITC onto the glass channels of the microchip was conducted to demonstrate the success of the immobilisation procedure before attempting it on the porphyrin species. The results of this test are shown in figure 3. Having successfully immobilised RBITC, the porphyrin was then immobilised using the same method. The porphyrinimmobilised chip was compared to batch and solution-phase methods described above for the ability to efficiently produce singlet oxygen, which was measured through the singlet oxygen-mediated oxidation of cholesterol. The results are shown in figure 4.
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Outlet
1 mm
Figure 3: RBITC successfully immobilised on the channel surfaces of the microfluidic chip using silanisation.
Figure 4: Graph showing the rate of conversion of cholesterol to oxidised products by singlet oxygen produced from porphyrin in (i) a batch procedure, (ii) free solution in continuous flow, and (iii) when immobilised on the channels of a microfluidic chip.
Rate of oxidation of cholesterol (μM/s)
Inlet
30 25 20 15 10 5 0 Batch
Flow
Immobilised
The batch reaction required 1 hour to complete and yielded the highest conversion of the three (50.9%). However, the time taken for the reaction showed that it was an inefficient process with a product conversion rate of 0.71 μM/s. The continuous flow reaction with the porphyrin in solution produced a higher rate of conversion (25 μM/s) than the batch procedure, but the highest conversion rate was achieved using the immobilised porphyrin method (29 μM/s), which also produced less variation in conversion rate. The higher rate of conversion for the immobilised porphyrin is explained by the porphyrin being in the path of the light throughout the experiment, as opposed to the solution-phase flow reaction in which the porphyrin was only in the light path for the solution’s residence time in the chip. The higher conversion rate and greater reproducibility of the immobilisation method suggests that this microfluidic technique is the most robust and consistent of the three procedures investigated. CONCLUSION We have demonstrated the ability to immobilise photosensitiser onto the glass channels of a microfluidic device, and proved that it is possible to produce singlet oxygen in an efficient manner. This could be used for safe and fast singlet oxygen-mediated oxidations for many reactions by reducing the need for large quantities of oxygenated solvents being stored and significantly speeding up these reactions. ACKNOWLEDGEMENTS The authors would like to thank the Clinical Biosciences Institute and the University of Hull for funding. REFERENCES [1] Clennan, E. L.; Pace, A., Advances in singlet oxygen chemistry, Tetrahedron 61, pp. 6665-6691 (2005). [2] Wootton, R. C. R.; Fortt, R.; De Mello, A. J., A microfabricated nanoreactor for safe, continuous generation and use of singlet oxygen, Org. Process Res. Dev., 6, pp 187-189 (2002). [3] Lier, J. E. V., Photobiological Techniques. Plenum Press: New York, (1991). CONTACT Ross W. Boyle, Tel: +44 (0) 1482 466353;
[email protected]
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