Chapter 31
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Fluorinated Amino Acids and Reagents in Protein Design and Biomolecule Separation 1
1
1,2,
He Meng , Venkateshwarlu Kalsani , and Krishna Kumar * 1
2
Department of Chemistry, Tufts University, Medford, M A 02155 Cancer Center, Tufts-New England Medical Center, Boston, M A 02110
The use of fluorinated amino acids and reagents has recently emerged as an important tool in bioorganic chemistry and protein design. The orthogonal phase properties of highly fluorinated compounds have been exploited in the design of protein ensembles that show enhanced chemical and thermal stability. Furthermore, highly fluorinated interfaces exhibit simultaneous hydrophobic and lipophobic behavior thus enabling their use in controlling helix-helix interactions within membranes. The insolubility of long chain fluorinated hydrocarbons has resulted in the development of reagents for use in peptide purification protocols and in proteomics using affinity separation. These applications along with the thermodynamic basis for phase separation of fluorinated materials have been highlighted here.
© 2007 American Chemical Society
In Current Fluoroorganic Chemistry; Soloshonok, V., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2007.
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488 The use of highly fluorinated compounds has stirred a mini revolution in organic synthesis and combinatorial chemistry. The use of highly fluorinated ("fluorous") solvents has been utilized in reaction acceleration (7), catalysis (2), combinatorial library synthesis and organic separation methodology (3,4). While fluorinated compounds are scarce in biology, on the other hand, phase separation of immiscible domains is ubiquitous, providing the driving force for formation of well-defined structures. Indeed, the structure of globular proteins, formation of the plasma membrane and sub-cellular organelles is essentially driven by the tendency of nonpolar substances to minimize their contacts with water. A new paradigm in supramolecular chemistry has been to make use of materials known to phase separate from water in the design of protein ensembles (5,6), for affinity purification in proteomics (4), and in the purification of biomolecules (7). The use of fluorous solvents and materials has primarily relied on the use of long chain perfluoroalkyl appendages tagged to the solvent, substrates and/or reagents. Recently, supramolecularly organized fluorinated surfaces have been used to direct protein folding and aggregation in aqueous solutions, and in the context of the nonpolar environments of biological membranes. These protein constructs differ in that the fluorinated groups are incorporated not in a contiguous covalent fashion, but upon folding of the peptide or protein, large expansive fluorinated surfaces are supramolecularly organized and displayed. Overall, this strategy provides the ability to control structural and functional properties of the resulting protein ensembles, and further endows them with extra-biological properties. In addition to use in protein design, fluorinated reagents have gained prominence in the purification of biomolecules synthesized using solid phase methods and in tagging and selective enrichment applications in proteomics.
Phase Separation of Fluorous Materials Perfluorocarbons phase separate from nonpolar organic solvents and water at room temperature. Hildebrand and Scott's theory of the solubility of nonelectrolytes (8) provides an empirical framework for estimating intermolecular interactions and propensity for mutual miscibility. On a simple level, a solubility parameter, 5, determines the extent to which two nonpolar liquids are miscible. The partial molal free energy (AF) of a component in a mixture of two nonpolar liquids is given by a sum of the entropy of mixing and the heat of mixing where x and x are the respective mole fractions, Vi and v are the molal volumes, q>\ and q>i are the volume fractions and 8\ and b\ are the solubility parameters (Equations 1 and 2). x
2
2
2
AF = RTlnx, +
2
AF = RT\nx + \ (o\ 2
2
2
-5 )
