Position-dependent impact of hexafluoroleucine and trifluoroisoleucine on protease digestion

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.


General information
All reactions were run under an argon atmosphere unless otherwise indicated. Room Coupling constants J are given in Hertz (Hz). Multiplicities are classified by the following abbreviations: s = singlet, d = doublet, t = triplet, q = quartet, br = broad or m = multiplet and combinations thereof. High resolution mass spectra were obtained on an Agilent ESI-ToF 6220 (Agilent Technologies, Santa Clara, CA, USA).

Peptide characterization
High resolution mass spectra were recorded on an Agilent 6220 ESI-ToF LC-MS spectrometer (Agilent Technologies Inc., Santa Clara, CA, USA) to identify the pure peptide products. The samples were dissolved in a 1:1 mixture of water and acetonitrile containing 0.1% (v/v) TFA and injected directly into the spray chamber by a syringe pump using a flow rate of 10 µL min −1 . A spray voltage of 3.5 kV was used, the drying gas glow rate was set to 5 L min −1 and the nebulizer to 30 psi. The gas temperature was 300 °C.
To verify purity of the synthesized peptides analytical HPLC was carried out on a Chromaster 600 bar DAD-System with CSM software (VWR/Hitachi, Darmstadt, Germany). The system works with a low-pressure gradient containing a HPLC-pump (5160) with a 6-channel solvent degasser, an organizer, an autosampler (5260) with a 100 µL sample loop, a column oven (5310) and a diode array flow detector (5430).
A LUNA TM C8 (2) column (5 μm, 250 × 4.6 mm, Phenomenex ® , Torrance, CA, USA) was used. As eluents water and ACN, both containing 0.1% (v/v) TFA were used, the flow rate was adjusted to 1 mL/min and the column was heated to 24 °C. The used gradient method is shown in Table S1. The UV-detection of the peptides occurred at 220 nm. The data were analyzed with EZChrom Elite software (version 3.3.2, Agilent Technologies, Santa Clara, CA, USA).   Figure S1: Analytical HPLC chromatograms of purified peptides; column: Luna TM C8 (5 µM, 250 × 4.6 mm, Phenomenex ® ); Solvent A was H2O, solvent B was acetonitrile, both containing 0.1% (v/v) TFA. The flow rate was 1 mL/min; linear gradient from 5% B to 70% B over 18 min (see Table S1).

Enzymatic digestion studies
Characterization of the enzymatic digestion reactions was carried out via analytical HPLC on a LaChrom-ELITE-HPLC-System from VWR International Hitachi (Darmstadt, Germany). The system contains an organizer, two HPLC-pumps (L-S7 2130) with solvent degasser, an autosampler (L-2200) with a 100 µL sample loop, a diode array flow detector (L-2455), a fluorescence detector (L-2485) and a high pressure gradient mixer. As eluents water and ACN, both containing 0.1% (v/v) TFA were used, and a flow rate of 3 mL/min was applied. The used linear gradients are shown in Table S3. For the non-fluorinated peptides method A was used to follow the digestion process, and for the fluorinated peptides method B was applied. For chromatograms where an insufficient baseline separation was observed, measurements were repeated using methods C [FA (pepsin), P2-LeuFA  S8 Figure  Identification of the proteolytic cleavage products (Table S4-S7) occurred according to the mass-to-charge ratios determined with an Agilent 6220 ESI-ToF-MS instrument (Agilent Technologies, Santa Clara, CA, USA). For this, the quenched peptide-enzyme-solutions after 120 min and 24 h incubation were analyzed. The solutions were injected directly into the spray chamber using a syringe pump with a flow rate of 10 µL min −1 . Spray voltage was set to 3.5 kV, a drying gas flow rate of 5 L min −1 was used, the nebulizer was set to 30 psi, and the gas temperature to 300 °C.
The fragmentor voltage was 200 V. Not all corresponding fragments could be detected.  Table S5: Identification of the cleavage products of the different peptides by ESI-ToF mass spectrometry after digestion with pepsin.