C-Terminal Arginine-Selective Cleavage of Peptides as a Method for Mimicking Carboxypeptidase B

C-Terminal residues play a pivotal role in dictating the structure and functions of proteins. Herein, we report a mild, efficient, chemoselective, and site-selective chemical method that allows for precise chemical proteolysis at C-terminal arginine dictated by 9,10-phenanthrenequinone independent of the remaining sequence. This biomimetic approach also exhibits the potential to synthesize C-terminal methyl ester (−CO2Me) peptides.


VI.
Supplementary Figure 1.Optimization of C-terminal Arginine Cleavage using peptide 1a General Procedure: Dansyl-HFANR-CONH 2 (1a) (1 mg, 1.14 µmol, 1 equiv.)was dissolved in 500 µL solvent H 2 O in a 1/2" dram vial.Next, 9,10-phenanthrenequinone (0.71 mg, 3.43 µmol, 3 equiv.)from a freshly prepared stock solution in ACN was added in one portion before 258 µL of solvent H 2 O was added to reach 900 µL total volume.For reaction utilizing organic bases (DMAP, DBU, or Et 3 N), 30 equiv.was added along with 100 µL of H 2 O yielding the final 1 mL volume.For reactions utilizing NaOH as the base, 0.8 M NaOH (100 µL) was added from a freshly prepared stock solution yielding a total volume of 1 mL with a concentration of 0.08 M NaOH.The concentration of the peptide in solution was 1.14 mM.The vial was stirred at 37 °C for 3 h.The solution was filtered via syringe filtration then analyzed via HPLC method A to determine the percent conversion to 2a.Anhydrous ACN DBU 0:100:0:0 a a DBU overlapped with peak for 2a making accurate percent conversion undeterminable.However, only minimal 1a is still seen at 11.6 min.

Entry
Please note: For optimized conditions using 0.08 N NaOH, HPLC method B was used for analysis therefore there is difference in the retention time of 1a and 2a in the HPLC traces.Retention Time (min) 11.9 9.2

9,10-phenanthrenequinone Degradation
Optimized procedure for cleavage of model peptide 1a (entry 7) 1 mg of Dansyl-HFANR-CONH 2 (1a, 1 equiv.)was dissolved in 500 µL H 2 O in a 1/2" dram vial.Next, 9,10-phenanthrenequinone (0.71 mg, 3.43 µmol, 3 equiv.)from a freshly prepared stock solution in ACN was added in one portion before 258 µL H 2 O was added to reach 900 µL total volume.Then 0.8 M NaOH (100 µL) was added from a freshly prepared stock solution yielding a total volume of 1 mL with a concentration of 0.08 M NaOH.The concentration of the peptide in solution was 1.14 mM.The vial was stirred at 37 °C for 3 h.The solution was filtered via syringe filtration then analyzed via HPLC method B to determine the percent conversion to 2a (>99%).Dansyl-HR-CONH 2 (10 mg, 1.8 µmol, 1 eq) was dissolved in 5 mL H 2 O in a 1.5" dram vial.Next, 9,10phenanthrenequinone (1.15 mg, 5.4 µmol, 3 equiv.)from a freshly prepared stock solution in ACN was added in one portion before 2.85 mL H 2 O was added to reach 9 mL total volume.Then 0.8 M NaOH (1 mL) was added from a freshly prepared stock solution yielding a total volume of 10 mL with 0.08 M NaOH.The concentration of the peptide in solution was 1.8 mM.The vial was stirred at 37 °C for 3 h.The solution was filtered via syringe filtration then analyzed via HPLC method B to determine the percent conversion to cleaved product Dansyl-H-CO 2 H (>99%). Fractions were combined and lyophilized revealing a fluffy white solid for characterization.152.9, 136.5, 135.4, 133.4, 131.3, 131.1, 130.6, 130.4, 128.9, 124.2, 120.7, 118.0, 116.4, 58.1, 45.8, 31.0.Dansyl-CHG-CO 2 H (1 mg, 1.82 µmol, 1 equiv.)was dissolved in 500 µL H 2 O in a 1/2" dram vial.Next, 9,10-phenanthrenequinone (1.14 mg, 5.47 µmol, 3 equiv.)from a freshly prepared stock solution in ACN was added in one portion before 172 µL H 2 O was added to reach 900 µL total volume.Then 0.8 M NaOH (100 µL) was added from a freshly prepared stock solution yielding a total volume of 1 mL with 0.08 M NaOH.The concentration of the peptide in solution was 1.91 mM.The vial was stirred at 37 °C for 24 h.The solution was filtered via syringe filtration then analyzed via HPLC method B. No modification occurred.Dansyl-KHG-CO 2 H (1 mg, 1.74 µmol, 1 equiv.)was dissolved in 500 µL H 2 O in a 1/2" dram vial.Next, 9,10-phenanthrenequinone (1.09 mg, 5.23 µmol, 3 equiv.)from a freshly prepared stock solution in ACN was added in one portion before 182 µL H 2 O was added to reach 900 µL total volume.Then 0.8 M NaOH (100 µL) was added from a freshly prepared stock solution yielding a total volume of 1 mL with 0.08 M NaOH.The concentration of the peptide in solution was 1.74 mM.The vial was stirred at 37 °C for 24 h.The solution was filtered via syringe filtration then analyzed via HPLC method B. No modification occurred.Dansyl-MHG-CO 2 H (1 mg, 1.74 µmol, 1 equiv.)was dissolved in 500 µL H 2 O in a 1/2" dram vial.Next, 9,10-phenanthrenequinone (1.08 mg, 5.21 µmol, 3 equiv.)from a freshly prepared stock solution in ACN was added in one portion before 184 µL H 2 O was added to reach 900 µL total volume.Then 0.8 M NaOH (100 µL) was added from a freshly prepared stock solution yielding a total volume of 1 mL with 0.08 M NaOH.The concentration of the peptide in solution was 1.74 mM.The vial was stirred at 37 °C for 24 h.The solution was filtered via syringe filtration then analyzed via HPLC method A. No modification occurred.Dansyl-SHG-CO 2 H (1 mg, 1.88 µmol, 1 equiv.)was dissolved in 500 µL H 2 O in a 1/2" dram vial.Next, 9,10-phenanthrenequinone (1.17 mg, 5.64 µmol, 3 equiv.)from a freshly prepared stock solution in ACN was added in one portion before 166 µL H 2 O was added to reach 900 µL total volume.Then 0.8 M NaOH (100 µL) was added from a freshly prepared stock solution yielding a total volume of 1 mL with 0.08 M NaOH.The concentration of the peptide in solution was 1.88 mM.The vial was stirred at 37 °C for 24 h.The solution was filtered via syringe filtration then analyzed via HPLC method B. No modification occurred.H 2 N-RHYK(dansyl)FA-CONH 2 (1 mg, 0.95 µmol, 1 equiv.)was dissolved in 500 µL H 2 O in a 1/2" dram vial.Next, 9,10-phenanthrenequinone (0.59 mg, 2.85 µmol, 3 equiv.)from a freshly prepared stock solution in ACN was added in one portion before 282 µL H 2 O was added to reach 900 µL total volume.Then 0.8 M NaOH (100 µL) was added from a freshly prepared stock solution yielding a total volume of 1 mL with a concentration of 0.08 M NaOH.The concentration of the peptide in solution was 0.95 mM.

OH
The vial was stirred at 37 °C for 3 h.The solution was filtered via syringe filtration then analyzed via HPLC method A to determine the percent conversion to imine product.Dansyl-RHQL-CONH 2 (1 mg, 1.27 µmol, 1 equiv.)was dissolved in 500 µL H 2 O in a 1/2" dram vial.Next, 9,10-phenanthrenequinone (0.80 mg, 3.82 µmol, 3 equiv.)from a freshly prepared stock solution in ACN was added in one portion before 240 µL H 2 O was added to reach 900 µL total volume.Then 0.8 M NaOH (100 µL) was added from a freshly prepared stock solution yielding a total volume of 1 mL with a concentration of 0.08 M NaOH.The concentration of the peptide in solution was 1.27 mM.The vial was stirred at 37 °C for 3 h.The solution was filtered via syringe filtration then analyzed via HPLC method A to determine the percent conversion to cleaved product.

Aldehyde formation of middle Arginine peptide
Dansyl-MEHFRWGKPV-CONH 2 (1 mg, 0.66 µmol, 1 equiv.)was dissolved in 500 µL H 2 O in a 1/2" dram vial.Next, 9,10-phenanthrenequinone (0.41 mg, 1.98 µmol, 3 equiv.)from a freshly prepared stock solution in ACN was added in one portion before 318 µL H 2 O was added to reach 900 µL total volume.Then 0.8 M NaOH (100 µL) was added from a freshly prepared stock solution yielding a total volume of 1 mL with a concentration of 0.08 M NaOH.The concentration of the peptide in solution was 0.66 mM.The vial was stirred at 37 °C for 3 h.The solution was filtered via syringe filtration then analyzed via HPLC method A to determine the percent conversion to cleaved product.Fractions were combined, lyophilized, and resubmitted to HPLC method B for characterization.

Procedure for cleavage of C+1 Histidine peptide 1c
Dansyl-HFAHR-CONH 2 (1c) (1 mg, 1.11 µmol, 1 equiv.)was dissolved in 500 µL H 2 O in a 1/2" dram vial.Next, 9,10-phenanthrenequinone (0.70 mg, 3.34 µmol, 3 equiv.)from a freshly prepared stock solution in ACN was added in one portion before 260 µL H 2 O was added to reach 900 µL total volume.Then 0.8 M NaOH (100 µL) was added from a freshly prepared stock solution yielding a total volume of 1 mL with a concentration of 0.08 M NaOH.The concentration of the peptide in solution was 1.11 mM.The vial was stirred at 37 °C for 3 h.The solution was filtered via syringe filtration then analyzed via HPLC method B to determine the percent conversion to 2c (>99%).Dansyl-HFASR-CONH 2 (1d) (1 mg, 1.18 µmol, 1 equiv.)was dissolved in 500 µL H 2 O in a 1/2" dram vial.Next, 9,10-phenanthrenequinone (0.74 mg, 3.54 µmol, 3 equiv.)from a freshly prepared stock solution in ACN was added in one portion before 252 µL H 2 O was added to reach 900 µL total volume.Then 0.8 M NaOH (100 µL) was added from a freshly prepared stock solution yielding a total volume of 1 mL with a concentration of 0.08 M NaOH.The concentration of the peptide in solution was 1.18 µM.The vial was stirred at 37 °C for 3 h.The solution was filtered via syringe filtration then analyzed via HPLC method B to determine the percent conversion to 2d (>99%).Dansyl-HFAVR-CONH 2 (1e) (1 mg, 1.16 µmol, 1 equiv.)was dissolved in 500 µL H 2 O in a 1/2" dram vial.Next, 9,10-phenanthrenequinone (0.73 mg, 3.49 µmol, 3 equiv.)from a freshly prepared stock solution in ACN was added in one portion before 255 µL H 2 O was added to reach 900 µL total volume.Then 0.8 M NaOH (100 µL) was added from a freshly prepared stock solution yielding a total volume of 1 mL with a concentration of 0.08 M NaOH.The concentration of the peptide in solution was 1.16 mM.The vial was stirred at 37 °C for 3 h.The solution was filtered via syringe filtration then analyzed via HPLC method B to determine the percent conversion to 2e (>99%).Dansyl-HFAWR-CONH 2 (1f) (1 mg, 1.06 µmol, 1 equiv.)was dissolved in 500 µL H 2 O in a 1/2" dram vial.Next, 9,10-phenanthrenequinone (0.66 mg, 3.17 µmol, 3 equiv.)from a freshly prepared stock solution in ACN was added in one portion before 268 µL H 2 O was added to reach 900 µL total volume.Then 0.8 M NaOH (100 µL) was added from a freshly prepared stock solution yielding a total volume of 1 mL with a concentration of 0.08 M NaOH.The concentration of the peptide in solution was 1.06 mM.The vial was stirred at 37 °C for 3 h.The solution was filtered via syringe filtration then analyzed via HPLC method A to determine the percent conversion to 2f (>99%).

peptide, Dansyl-WHG-CO 2 H
Dansyl-WHG-CO 2 H (1 mg, 1.58 µmol, 1 equiv.)was dissolved in 500 µL H 2 O in a 1/2" dram vial.Next, 9,10-phenanthrenequinone (1.0 mg, 4.75 µmol, 3 equiv.)from a freshly prepared stock solution in ACN was added in one portion before 200 µL H 2 O was added to reach 900 µL total volume.Then 0.8 M NaOH (100 µL) was added from a freshly prepared stock solution yielding a total volume of 1 mL with 0.08 M NaOH.The concentration of the peptide in solution was 1.58 mM.The vial was stirred at 37 °C for 24 h.The solution was filtered via syringe filtration then analyzed via HPLC method A. No modification occurred.