Position-dependent impact of hexafluoroleucine and trifluoroisoleucine on protease digestion

Abstract

Rapid digestion by proteases limits the application of peptides as therapeutics. One strategy to increase the proteolytic stability of peptides is the modification with fluorinated amino acids. This study presents a systematic investigation of the effects of fluorinated leucine and isoleucine derivatives on the proteolytic stability of a peptide that was designed to comprise substrate specificities of different proteases. Therefore, leucine, isoleucine, and their side-chain fluorinated variants were site-specifically incorporated at different positions of this peptide resulting in a library of 13 distinct peptides. The stability of these peptides towards proteolysis by α-chymotrypsin, pepsin, proteinase K, and elastase was studied, and this process was followed by an FL-RP-HPLC assay in combination with mass spectrometry. In a few cases, we observed an exceptional increase in proteolytic stability upon introduction of the fluorine substituents. The opposite phenomenon was observed in other cases, and this may be explained by specific interactions of fluorinated residues with the respective enzyme binding sites. Noteworthy is that 5,5,5-trifluoroisoleucine is able to significantly protect peptides from proteolysis by all enzymes included in this study when positioned N-terminal to the cleavage site. These results provide valuable information for the application of fluorinated amino acids in the design of proteolytically stable peptide-based pharmaceuticals.

Keywords: fluorinated amino acids; hexafluoroleucine; peptide drugs; protease stability; trifluoroisoleucine.

Figure 1

(a) Structures of isoleucine (1), leucine (2), and their fluorinated analogues 5,5,5-trifluoroisoleucine (3, TfIle) and 5,5,5,5’,5’,5’-hexafluoroleucine (4, HfLeu). The van der Waals volumes given in parentheses correspond to the side chains (starting at α-carbon), and were calculated according to Zhao et al. [43]; (b) Names and amino acid sequences of the studied peptides; the substitution positions are marked as Xaa; Xaa = Leu, HfLeu, Ile or TfIle. Positions are named according to Schechter and Berger nomenclature [41].

Figure 2

Percentage of substrate remaining after incubation for 120 min (left) and 24 h (right) with α-chymotrypsin in 10 mM phosphate buffer, pH 7.4, at 30 °C. The data shown represent the average of three independent measurements. Errors are derived from the standard deviation.

Figure 3

Cleavage positions observed in the digestion of library peptides with α-chymotrypsin.

Figure 4

Percentage of substrate remaining after incubation for 120 min (left) and 24 h (right) with pepsin in 10 mM acetate buffer, pH 4.0, at 30 °C. The data shown represent the average of three independent measurements. Errors are derived from the standard deviation.

Figure 5

Cleavage positions for digestion of the different peptides with pepsin.

Figure 6

Percentage of substrate remaining after incubation for 120 min (left) and 24 h (right) with elastase in 100 mM Tris/HCl buffer, pH 8.4, at 37 °C. The data shown represent the mean of three independent measurements. Errors are derived from the standard deviation.

Figure 7

Cleavage positions for digestion of the different peptides with elastase.

Figure 8

Percentage of substrate remaining after incubation for 120 min (left) and 24 h (right) with proteinase K in 50 mM Tris/HCl buffer, containing 10 mM CaCl₂ pH 7.5, at 37 °C. The data shown represent the mean of three independent measurements. Errors are derived from the standard deviation.

Figure 9

Cleavage positions found with proteinase K.

Figure 10

Dimension of stabilization or destabilization upon TfIle or HfLeu incorporation compared to the non-fluorinated analogues containing Ile or Leu, respectively, for all four different enzymes studied here and measured after 24 h of incubation.