Modification of Peptides and Proteins with Fluorinated Amino Acids

One other aspect of peptide and protein research that we devote our efforts to is expanding the toolkit of amino acid building blocks to improve the pharmacokinetic and physicochemical profiles of peptide-based drugs and materials, respectively. Amino acid residues carrying functional groups that are orthogonal to biological systems may serve as either biophysical probes for the detailed determination of structure-activity relationships, or components of tailor-made biomolecules. In particular, the judicious incorporation of fluorine atoms has been shown to enhance protein stability in vivo and to improve the physical properties of protein-based materials. We have developed peptide-based models to systematically studying the complex molecular interactions of fluoroalkyl groups within native polypeptides regarding space filling, lipophilicity, and hydrogen-bonding. Our peptide models are sensitive enough to report on changes that are brought about by the introduction of just one fluorine atom. We were able to determine how fluorinated amino acids influence the structure and thermodynamic stability of the α-helical coiled coil (Salwiczek, ChemBioChem, 2009). Furthermore, the application of methods such as surface plasmon resonance (SPR) and phage display technology enables detailed study of the binding between natural and nonnatural peptide partners (Vagt, Org. Biomol. Chem., 2010). These studies will pave the way for optimizing the incorporation of fluorinated amino acids for the de novo design of peptide-based materials and drugs. 
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Analogues of (S)-2-aminobutyric acid (Abu) with increasing fluorine content have been extensively characterized (Samsonov, Salwiczek, et al., J. Phys. Chem. B, 2009). Interestingly, whereas a single carbon-fluorine bond in the side chain of Abu decreases the hydrophobicity of the residue, a significant and additive increase in hydrophobicity is observed with the introduction of further carbon-fluorine bonds. Thus, although MfeGly is larger in volume than Abu, it is less hydrophobic. DfeGly is also less hydrophobic than its hydrocarbon/fluorocarbon surface area would suggest. DfpGly, which is very close in size to leucine, is even more polar than the much smaller valine.

Figure 3. (a) Chemical structure of Abu-analogues with increasing fluorine content . (b) Correlation of retention time and solvent accessible surface area of amino acids of side chain fluorinated amino acids. (c) Packing of a and d positions in a parallel coiled coil dimer. (d) Bar chart depicting α-helix propensity vs. hydrophobicity.

 

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Using a coiled coil heterodimeric model system and substitutions focused to the hydrophobic core, we have shown that the impact of size and polarity of the fluorinated building blocks highly depends upon the immediate environment of the substitution. Coiled coils are α-helical assemblies that possess a (pseudo-) repetitive primary sequence (abcdefg)n, in which hydrophobic side chains generally occupy the a- and d-positions (Figure 3). At central a positions, where the Cα-Cβ bond vectors point out of the hydrophobic core and into solution, stability is mainly determined by side chain volume. At central d positions, however, the destabilizing impact of fluorine-induced polarity prevails, since here the Cα-Cβ bond vectors point directly into the hydrophobic core (Salwiczek, Chem. Eur. J., 2009).

To efficiently screen for preferred interaction partners of the four aminobutyric acid analogues, phage display was carried out (Vagt, Org. Biomol. Chem., 2010). The predetermined secondary and tertiary structure of coiled coils are the features of our system that allow this technology to be exploited. Thus, fluorinated amino acids are incorporated into the hydrophobic positions of one strand of the heterodimer, and the residues of the other strand expected to directly interact with the nonnatural analogues are randomized to create a library that is displayed on bacteriophage. Despite the polarity that is induced by partial fluorination, the interaction profiles of all four fluoroalkylated amino acids studied so far are similar to that of aminobutyric acid. Moreover, independent of the respective microenvironment, incorporating fluorinated aminobutyric acids into a central a- or d-position of a parallel heterodimeric coiled coil leads to pairing characteristics that are similar to their canonical analogues (Nyakatura, RSC Advances, 2013).

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Fluorinated amino acids have been rarely investigated in amyloid forming peptides. This is surprising since they generally show relatively low helix propensities in comparison to their native counterparts and may thus be more suitable for the incorporation into beta sheet rich structures. In order to elucidate the impact of amino acid side chain fluorination on amyloid formation, we have substituted valine residues within solvent exposed regions of a de novo designed coiled coil peptide that is able to undergo a conformational switch to beta-sheet rich amyloid fibrils. We found that these valine residues are responsible for the structural rearrangement, and resolved the internal architecture of peptide strands within the fibrils (Gerling, Biomacromol., 2011). Interestingly, the number of carbon-fluorine bonds present within the side chain has a significant effect on the kinetics of the structural transition into amyloids. Increasing the fluorine content increases the rate of folding. This effect can be explained by the interplay of several factors. Although size and hydrophobicity directly and additively increase with fluorination, α-helical propensity decreases. Thus, the incorporation of fluorinated building blocks into amyloidogenic structures could be of use in materials science applications.  

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A full characterization of proteolytic stability is critical for the successful application of fluorinated peptide based pharmaceuticals. To investigate the impact of fluoroalkyl substitution on the protease resistance of peptides, a small model peptide library was synthesized in which Abu, DfeGly, and TfeGly are incorporated at different guest positions. When subjected to proteolysis, these model peptides allow for a systematic comparison of the substitution effects with respect to the kind of amino acid as well as its position relative to the enzymatic cleavage site (Asante, Bioorg. Med. Chem., 2013). In general, the impact of side chain fluorination on the stability towards α-chymotrypsin and pepsin digestion appears to be a complex phenomenon which depends not only on the position but also on the stiochiometry of fluorination, as well as on the interactions with the enzyme subsites. 

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In order to facilitate the site specific incorporation of fluorinated amino acids into proteins, two methods are currently applied in our group, (i) in vitro non-sense suppression based protein expression and (ii) total chemical synthesis. In the first approach, an amber stop codon is introduced into a desired position in the protein encoding DNA sequence, and a fluorinated amino acid is chemically coupled to a suppressor tRNA (Ye, Beilstein J. Org. Chem., 2010). Subsequently, protein expression is carried out by means of a cell-free translation system. In the second approach, peptide fragments are synthesized by solid phase peptide synthesis, and the full-length protein is generated by means of native chemical ligation. To investigate fluorine induced effects in the context of protease-inhibitor interactions, our group is currently investigating the basic bovine pancreatic trypsin inhibitor (BPTI) and various proteases. When substituted at a site of the inhibitor that directly interacts with chymotrypsin, DfeGly increases protein stability and preserves inhibition. 

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1. In addition to the above mentioned in vitro based approaches, the Koksch group is also working on developing a method for the in vivo synthesis of ‘fluorous proteins’ i.e. proteins with a high content of fluorinated amino acids. This includes the design of an expression system in which increased cytosolic concentrations of the relevant aminoacyl-tRNA synthetases enhance the residue specific incorporation of fluorous building blocks. These studies are being carried out in collaboration with the Budisa group of TU Berlin, and are expected to pave the way for the use of the beneficial properties of fluorinated amino acids for a deliberate de novo design of biologically relevant peptide drugs and fluorinated protein-based materials.


2. One major goal in the development of peptide-based drugs is to overcome the intrinsic protease-susceptibility of natural peptides, which limits their clinical use. In this context, one of the newer research projects in the Koksch group focuses on the generation of peptide-based inhibitors of Candida albicans secreted aspartic acid proteases (SAPs) (Cadicamo, Biochem. Pharmacol., 2013).

Figure 4. Model of peptide inhibitor – SAP3 interaction.

SAPs are important virulence factors of C. albicans, which is the most common fungal pathogen in humans. Fluorinated building blocks could considerably extend the repertoire of functionalities that can be used to enhance the selectivity of current-generation inhibitors, with respect to SAPs of the human host.

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