Jordan H. Wosnick Outline of Proposed Research

Jordan H. Wosnick – Outline of Proposed Research – Page 1 of 6

Jordan H. Wosnick – Outline of Proposed Research

Introduction

Control over the structure of matter and flow of energy lies at the heart of the chemical sciences. My primary interests lie in the development of materials and techniques that show specific or unusual interactions with other molecules, with energy, or with living cells. In Project 1, I describe the use of an unusual class of polyatomic anions – the polyoxometalates – as a platform for the construction of highly efficient fluorescence-quenching groups. These materials will be useful in the construction of beacons and probes for molecules of biological interest. Project 2 focuses on the development of new methodologies in microwave-heated organic synthesis, using interactions with highly microwave-absorbing materials to achieve selective heating of tethered molecules. Finally, Project 3 describes a technique for the rapid construction of arbitrarily patterned, cell-adhesive materials using photolithography followed by polyelectrolyte deposition.

All three of these projects are highly interdisciplinary, and together they draw from the fields of synthetic chemistry, materials science, physical chemistry, biomaterials and cell biology. I have presented each project in terms of a series of initial demonstrations followed by a number of key research questions. As such, there are facets of this research that would provide broad training for either undergraduates or graduate students. My research background in organic materials chemistry, fluorescence spectroscopy and microscopy, bioconjugate chemistry and biomaterials gives me both the unique vantage point and the technical skills required for these projects.

Project 1. Polyoxometalates and their derivatives as fluorescence quenchers for supramolecular and biological sensory schemes

Background

The phenomenon of fluorescence resonance energy transfer (FRET) is extensively used in the design of fluorescent assays for nucleic acids (molecular beacons) and enzymes (fluorogenic probes). FRET between donor and acceptor groups is a complex phenomenon that depends on the fluorescence quantum yield of the donor, the extinction coefficient of the acceptor, the distance and relative orientation of the two chromophores, and the spectral overlap between the donor fluorescence and the acceptor absorbance.[1] While the optimization of FRET from the ‘donor side’ (maximization of fluorophore quantum yield) has achieved a great deal of attention, an alternate approach – the optimization of quenching properties in FRET acceptors – remains in its infancy, with only a few ill-defined or proprietary examples (gold nanoparticles, QSY and Black Hole Quenchers).

In this project, I propose to investigate polyoxometalates (POMs) as a platform for the development of potent fluorescence quenchers that have inherent biological and supramolecular capabilities. POMs are discrete, well-defined metal-oxide clusters that have drawn considerable interest for both structural and functional reasons.[2] They are potent electron acceptors and have been studied extensively in the form of charge-transfer crystalline materials with a variety of organic donor moieties.[3] Some types of POM have also been shown to have unexpected biological properties, including anti-tumour activity and potent inhibition of protein activity (e.g. HIV protease and FGF-2).[4] Over the last several years, a number of research groups have developed methods for the covalent functionalization of POMs with organic ligands.[5] In particular, detailed procedures have been devised for the regioselective modification of the so-called Lindquist-type POMs (e.g. [Mo6O19]2-, Figure 1.1) with arylimido ligands.[6] Based on these protocols, Zhang and co-workers have synthesized a number of conjugated poly(phenylene ethynylene)s (PPEs) containing POMs embedded in the main chain of the polymer or attached to the polymer backbone.[7] They reported efficient fluorescence quenching viacharge transfer in cases where the POM was joined to the PPE through a π-conjugated linkage, but did not otherwise describe the quenching behaviour of these species.

Project goals

This project will explore the scope and utility of POM fluorescence quenching for applications in fluorogenic assay development and supramolecular host-guest chemistry. Preliminary experiments will seek to establish the quenching behaviour of the model POM [Mo6O19]2- toward small-molecule fluorophores (Stern-Volmer constants, static vs.dynamic quenching). Following this, specific research applications will be addressed as follows:

• Covalent functionalization of POMs can significantly change their absorption properties and also affect the shape and charge distribution of the POM surface. What effects do these changes have on fluorophore quenching? Lindquist-type POMs such as [Mo6O19]2- have absorption profiles that match well with the emission of coumarin fluorophores commonly used in fluorogenic probes. Can covalently tethered POMs be used as fluorescence quenchers in these environments (Figure 1.2)?
• POMs bind to proteins through a combination of shape and charge complementarity. Do the steric and electronic changes that result from non-covalent binding of POMs to biological targets have a significant effect on fluorescence quenching? Can these effects be used to construct fluorescent ‘turn-on’ or ‘turn-off’ beacons or probes for these targets?
• Kochi has demonstrated the formation of charge-transfer complexes in crystals of POM acceptors with aminoalkyl-substituted polycyclic aromatic hydrocarbons.3 Can this effect be replicated in solution using a macromolecular host based on a functionalized calix[n]arene (n = 4, 6) or related macrocycle (Figure 1.3)?

Future research directions in this project will be directed toward the application of POMs in ‘super-quenching’ chromophores for long-range FRET and fluorogenic probe applications.

Project 2. Charged microparticle immobilization for selective microwave dielectric heating of reaction mixtures

Background

Microwave heating for organic synthesis – an increasingly common technique in both academic and industrial labs – can greatly reduce the amount of time required to perform typical reactions, resulting in higher yields and more rapid synthetic procedures.[8] Liquid samples in a microwave oven are heated by the direct coupling of microwaves with permanent dipoles in the sample. The energy of the impinging microwaves is partially dissipated as the dipoles attempt to ‘follow’ the oscillating electric field, resulting in sample heating. For this reason, substances with strong dipoles are generally heated more efficiently under microwave irradiation. A second heating mechanism operates in electrolytes, where the translational motion of ions themselves contributes to solution heating. This effect is strong enough to make ordinary tap water heat more quickly than distilled water in a microwave oven.[9]

Dissolved microwave-absorbing substances, such as ionic liquids, can increase the microwave-induced heating rate of non-polar solvents dramatically.[10] Related observations have been made using heterogeneous reaction systems. For example, Bednarz et al. investigated microwave-mediated oxidation reactions using Magtrieve, a solid form of CrO2,and observed specific coupling of microwave energy to the ferromagnetic material.[11] Very recently, the Strouse group reported the preparation of semiconductor ‘quantum dots’ of improved quality using microwave heating, a phenomenon they ascribed to selective coupling of microwaves to polar species.[12] In related work, Jacobson and co-workers found that DNA duplexes conjugated to gold nanoparticles could be selectively denatured in an oscillating magnetic field. In this experiment, DNA that was not directly attached to the nanoparticles remained stable.[13]

The ability to selectively activate and ‘address’ a particular species in a reaction mixture would be of tremendous utility in organic synthesis, enabling the researcher to conduct multiple, normally incompatible reactions in sequence without intervening purification steps – a form of site-isolation.[14]In this project, I propose to use the phenomenon of selective dielectric coupling to microwave-absorbing species to achieve the directed energetic addressing of particular moieties in a reaction mixture. This application-focussed research will make use of commercially available microwave reactors to expand the scope of rapid, ‘one-pot’ organic synthesis.

Project goals

This project will explore the use of reactant immobilization on charged microparticles to promote particular organic reactions in a microwave reactor. Initial investigations will make use of alternating polyelectrolyte layer-by-layer (LbL)[15] assembly to fashion microparticles of defined size and zeta-potential. A portfolio of charged particles will be created by depositing layers of common polycations (polydimethyldiallylammonium chloride and polyallylamine will be used initially) on the surface of commercially available, monodisperse silica microspheres (~ 0.5 to 5 μm – Figure 2.1).[16] Specific questions to be addressed in this project are as follows:

• In what way does the heating rate of microparticle suspensions depend on microparticle size, surface charge, layer density (variable through changes in ionic strength), and microparticle concentration?
• Microparticle immobilization of a reactant should show the greatest advantage in solvents that are microwave-transparent. Do microparticles provide larger heating rate enhancements for solvents with small permanent dipole moments (hydrocarbons, dichloromethane, dioxane)?
• Microparticle immobilization studies will be carried out using photolytically cleavable linker groups commonly used in solid-phase organic synthesis.[17] A simple intramolecular Michael-type cyclization reaction[18] (Figure 2.2) will be used to quantify the preferential activation of microparticle-immobilized reactants. A series of careful control experiments will be required to determine the extent of activation provided by immobilization on the charged microparticle. Does microparticle immobilization provide a rate enhancement versusan electronically similar reaction (e.g., one in which R is different – Figure 2.2) occurring in the same solution? Is any rate enhancement for the immobilized reactant observed when the reaction mixture is heated conventionally? Is any rate enhancement observed when microparticles and reactants are simply mixed (not covalently linked)?
• Some microwave reactors allow reaction mixtures to be cooled by a jet of compressed air during microwave irradiation. In a system where the solvent is microwave-transparent, this will allow for even greater dissipation of energy in microwave-absorbing species without raising the bulk temperature of the solution. Does simultaneous cooling provide an advantage for microparticle-immobilized reactions?

Future directions for this work may include developing systems in which rapid, specific immobilization to a polyelectrolyte support can take place in situ during a reaction sequence, without further purification. This may take the form of microparticles that can ‘capture’ specific products via a ‘click’-type azide-alkyne cycloaddition on addition to a reaction mixture. Subsequent irradiation in a microwave reactor would then lead to preferential activation of the immobilized species. Such procedures will facilitate one-pot synthetic methodologies, and have the potential to make a significant impact on the speed and facility of microwave-promoted organic synthesis.

Project 3. Combined photolithography and polyelectrolyte layer-by-layer deposition for the production of patterned, cell-adhesive substrates

Background

Rapid advances in biomaterials and tissue-engineering research in recent years have been prompted by an increased understanding of how cells respond to the chemical properties of their environment. One manifestation of this progress is the production of materials that spatially direct cell adhesion – an achievement that opens the door to the in vitro production of biomimetic ‘tissue’.[19] However, it has been recognized that the control of cell adhesion is only one aspect of the mechanism that organisms use in tissue development. For example, neurons in injured nerve tissue have been shown to respond to chemotactic gradientsof soluble neurotrophic factors (usually small proteins) that guide severed axons back to their original targets.[20] This phenomenon has also been examined in the laboratory, where concentration gradients of both the haptotactic(cell-adhesive) peptide fragment IKVAV[21] and the nerve growth factor NGF[22] have been used to control the direction of neurite extension from cells in model systems. However, differing methods of immobilization – including the use of gradient makers, photolithography, and microfluidic techniques – make it difficult to compare results between groups and to quickly prepare highly reproducible gradients of chemotactic or haptotactic cues. Photochemical techniques used to create gradients often function either by weakly immobilizing a pre-established gradient in a photo-crosslinked matrix[23] or by non-specifically coupling proteins to a hydrocarbon-based support (such as a polystyrene culture dish).[24]

A full understanding of the factors that affect tissue regeneration, especially the complex sequence of events that occurs during nerve repair, will require a versatile platform that can be used to prepare cell culture ‘test beds’ for the study of cell responses to both immobilized and soluble cues. In this project, I propose to use a combination of directed photolithography and alternating polyelectrolyte layer-by-layer (LbL) deposition14 to prepare patterned, cell-adhesive substrates with gradient and drug-release capabilities. These platforms will be used to study cell migration and neurite extension in a controlled, pre-defined environment.

Project goals

The LbL deposition technique, in which charge overcompensation is used to deposit alternating layers of polycations and polyanions, is a powerful tool for creating polymer microstructures in a rapid and reproducible manner.15 In initial stages of this work, glass slides will be covalently modified with a polyethylene glycol-modified (PEGylated) photocleavable protecting group that unveils free amine functionality when irradiated with ultraviolet light, thus creating an area of positive charge at physiological pH. A related approach has been used by Mrksich[25] to pattern alkanethiolates on gold surfaces. In the present application, a non-adhesive ‘cell-resist’ will be prepared using a brominated hydroxycoumarin carbamate, a non-toxic amine photocaging group with a high quantum efficiency.[26] Substrates will be mounted on an X-Y translation stage and moved under the focal point of a UV light source or laser (~ 370 nm). Photodeprotection of amine groups will result in a local positive charge over irradiated regions, allowing the surface deposition of polyelectrolytes (polystyrene sulfonate and polyallylamine will be used initially) by the LbL method (Figure 3.1).[27] Confirmation of selective deposition will be carried out by fluorescence microscopy of substrates coated with dye-labelled polyelectrolytes. The ability to selectively deposit charged biomolecules will be tested using fluorescently labeled lysozyme (pI ~ 10) and protein G (pI ~ 4.5). Furthermore, the ability to selectively deposit two different polyelectrolytes (by repeated irradiation-deposition cycles) will be tested by similar methods.

Following these initial proof-of-principle demonstrations, the following specific research questions will be addressed:

• The naturally occurring polysaccharide chitosan is a moderate promoter of cell adhesion, while synthetic polycations such as polylysine can be modified with laminin- and fibronectin-derived cell-adhesive peptides such as GRGDS and IKVAV.[28] What is the behaviour of PC-12 cells (a tumour-derived cell line that shows nerve cell phenotypes) when presented with a substrate that is patterned with regions of receptor-specific and non-specific cell adhesion?
• The coumarin caging group proposed (Figure 3.1) for the ‘cell-resist’ can be deprotected with longer wavelengths and shorter exposures relative to other photocaging groups (e.g. nitroveratryl). Can repeated irradiation-deposition cycles be performed in situ, on the translation stage, to pattern different polycations on the substrate surface (Figure 3.2) without causing degradation?
• Gradients of haptotactic cues are important in guiding neurite extension. Can changes in scan rate or UV light intensity be used to create microscale gradients of local positive charge on the substrate surface, and can these be used to electrostatically assemble gradients of cell-adhesive polycations?
• Cells may respond to gradients of both haptotactic and chemotactic cues at the same time. Hammond has recently demonstrated controlled, pseudo-first-order release behaviour from LbL films.[29] Can regions containing electrostatically immobilized chemotactic cues (such as NGF) be patterned alongside (or at the end of) gradients of haptotactic cues, such as the peptide GRGDS? What is the response of PC-12 cells to these substrates?

Future directions in this project will involve the creation of directed ‘circuits’ of cells, guided by patterned cell adhesion and micro-gradients. Such systems would prove invaluable in the study of cell-cell interactions and tissue formation and have the potential to contribute to fundamental understandings in cell biology.

References

[1] Described in detail in Lakowicz, J. R. Principles of fluorescence spectroscopy, 2nd ed. Kluwer Academic, 1999.

[2] Books and reviews: (a) Pope, M. T.; Mueller, A., eds. Polyoxometalates: from platonic solids to anti-retroviral activity. Kluwer Academic, 1994. (b) Pope, M. T.; Mueller, A., eds. Polyoxometalate chemistry from topology via self-assembly applications. Kluwer Academic, 2001. (c) Chem. Rev. 1998, January issue (devoted entirely to POMs).

[3] Le Maguerès, P.; Hubig, S. M.; Lindeman, S. V.; Veya, P.; Kochi, J. K. J. Am. Chem. Soc. 2000, 122, 10073.

[4] Reviews: (a) Hasenknopf, B. Front. Biosci. 2005, 10, 275. (b) Rhule, J. T.; Hill, C. L.; Judd, D. A.; Schinazi, R. F. Chem. Rev. 1998, 98, 327.

[5] Peng, Z. Angew. Chem. Int. Ed. 2004, 43, 930.

[6] (a) Xu, L.; Lu, M.; Xu, B.; Wei, Y.; Peng, Z.; Powell, D. R. Agnew. Chem. Int. Ed. 2002, 41, 4129. (b) Wei, Y.; Xu, B.; Barnes, C. L.; Peng, Z. J. Am. Chem. Soc. 2001, 123, 4083. (c) Kang, J.; Xu, B.; Peng, Z.; Zhu, X.; Wei, Y.; Powell, D. R. Agnew. Chem. Int. Ed.2005, 44, 6902.

[7] (a) Lu, M.; Xie, B.; Kang, J.; Chen, F.-C.; Yang, Y.; Peng, Z. Chem. Mater. 2005, 17, 402. (b) Xu, B.; Lu, M.; Kang, J.; Wang, D.; Brown, J.; Peng, Z. Chem. Mater. 2005, 17, 2841.

[8] (a) Tierney, J. P.; Lidström, P., eds. Microwave assisted organic synthesis. Blackwell/CRC Press, 2005. (b) Loupy, A., ed. Microwaves in organic synthesis. Wiley-VCH, 2002. (c) Kappe, C. O. Angew. Chem. Int. Ed. 2004, 43, 6250.

[9] Lidström, P.; Tierney, J.; Wathey, B.; Westman, J. Tetrahedron 2001, 57, 9225.

[10] This should be a ref about the doping of a rxn mixt with RTILs to boost heating rates.

[11] Bogdal, D.; Lukasiewicz, M.; Pielichowski, J.; Miciak, A.; Bednarz, Sz. Tetrahedron 2003, 59, 649.