Selective N-terminal functionalization of native peptides and proteins

A highly site-selective modification of peptides/proteins with aldehydes or carbohydrates under mild conditions was achieved.


General Information: Chemical and Protein Materials.
Benzaldhyde, n-propionaldhyde, p-acetylbenzaldehyde, p-ethynylbenzaldehyde, 2pyridinecarboxyaldehyde, glucose, Maltose, sodium cyanoborohydride, Fmoc N-hydroxysuccinimide ester, RNase A, Lysozyme were purchase from Sigma-Aldrich and used without purification. Insulin was purchased from Life Technologies Co. Model peptides and GLP-1 were synthesized via Fmoc solid phase peptide synthesis. Dithiothreitol (DTT) was purchased from Gold Bio Technology. Fmoc-protected amino acids were purchased from Protein Technologies Inc.

HPLC, LC/MS and Mass Spectrometry
Preparative reverse-phase HPLC of crude peptides was performed on Luna 5u C8 100 Å (250 ×10 mm) at 3 mL/min with a water/acetonitrile gradient in 0.1% TFA on an Agilent 1260 HPLC system. Fractions collected from preparative were analyzed by LC/MS on a XBridge C18 5-μm (50 × 2.1 mm) column at 0.4 mL/min with a water/acetonitrile gradient in 0.1% formic acid on an Agilent 6120 Quadrupole LC/MS system. Fractions containing targeted product (based on LC/MS) were collected and lyophilized.
Protein analyses and peptides LC-MS/MS analyses were performed using a hybrid Q-TOF mass spectrometer (Bruker, Maxis ETD-II) equipped with Eksigent Nano-LC 400 system (Captive Spray source for peptides; Microspray source for big protein). The trypsin digested peptide fragments chromatography was performed using Waters Atlantis c18 nano column (0.1 mm × 100 mm size for peptides; 0.3 mm × 100 mm size for proteins) with a CH 3 CN/ dd H 2 O gradient mobile phase containing 0.1% formic acid (flow rate: 0.4 µL/min for peptides; 5 µL/min for big protein).

Peptide Synthesis
Peptides XYSKEASAL (X = 20 natural amino acids) and GLP-1 were synthesized via Fmoc solid phase peptide synthesis on a commercial peptide synthesizer (Alstra; Biotage, Inc). Automated peptide synthesis was carried out in a 10 mL reactor vial with the following protocols (for 0.1 mmol scale). For Fmoc deprotection: (i) 4.5 mL of 20% piperidine in DMF; (ii) mix 2 × 3 min (new solvent delivered for each mixing cycle). For amino acid coupling: (i) 1.25 mL of 0.4 M Fmoc-protected amino acid in DMF; (ii) 1.225 mL of 0.4 M HBTU or HATU (HBTU and Rink Amide MBHA resin HL was used for peptides XYSKEASAL [X = 20 natural amino acids]; HATU and H-Rink Amide ChemMatrix for GLP-1) in DMF; (iii) 1.0 mL of 1.0 M DIPEA in DMF; and (iv) mix for 5 min at 75 o C (for cysteine and histidine coupling: mix for 10 min at 50 o C; for Arginine coupling: mix for 15 min at 50 o C and coupling twice). For DMF washing (performed between deprotection and coupling steps): (i) 4.5 mL of DMF; (ii) mix 45 s. Upon completion of the peptide chain, resins were washed with DCM and dried (using vacuum) for 20 min. Then peptide was cleaved from the resin by exposure to cleavage cocktail for 2.5 h, which were prepared with 12.5 mL TFA, 330 μL water, 330 μL TIS. The peptide was precipitated with ethyl ether at 4 o C, followed by HPLC purification and lyophilization.
3. General procedure for the reductive amination between peptide/protein and aldehyde/carbohydrate For model peptide reacted with aldehyde: The mixture of 1 (2.7 µmol) and NaBH 3 CN (5 equiv, 13.5 µmol, 1.0 mg) were dissolved in 300 µL citric acid buffer (pH = 6.1). 0.5 M aldehyde/DMSO solution (2 equiv, 5.4 µmol, 13 µL) was added into the system and kept stirring for 4-6 hours at room temperature. The reaction solution was diluted with distilled deionized water (2.0 mL) and subjected to preparative reversephase HPLC (Agilent 1260 HPLC system). The corresponding liquid fractions were collected and lyophilized.

For model peptide reacted with carbohydrate:
The mixture of 1a (2.7 µmol), carbohydrate (10 equiv, 27 µmol) and NaBH 3 CN (5 equiv, 13.5 µmol, 1.0 mg) were dissolved in 300 µL acetic acid buffer (pH = 6.0) and incubated at 37 o C (Incubate 20 hours for the reaction with maltose; 2 days for the reaction with glucose). The reaction solution was diluted with distilled deionized water (2.0 mL) and subjected to preparative reverse-phase HPLC (Agilent 1260 HPLC system). The corresponding liquid fractions were collected and lyophilized. For proteins reacted with benzaldehyde: The mixture of protein (3 mg), NaBH 3 CN (5 equiv) was dissolved in 300 µL citric acid buffer (pH = 6.1). 0.5 M benzaldehyde/DMSO solution (2 equiv) was added into the system and kept stirring at room temperature. (3 hours for GLP-1; 6 hours for insulin; 24 hours for RNase; 48 hours for lysozyme; 10 hours for aldolase and phosphokinase). The reaction solution was diluted with distilled deionized water (2.0 mL) and subjected to preparative reverse-phase HPLC (Agilent 1260 HPLC system). The corresponding liquid fractions were collected and lyophilized.

S4
The mixture of insulin (6 mg), NaBH 3 CN (5 equiv) were dissolved in 300 µL citric acid buffer (pH = 6.1). 0.5 M 4-ethynylbenzaldehyde/DMSO solution (2 equiv) was added into the system and kept stirring at room temperature for 6 hours. The reaction solution was diluted with distilled deionized water (2.0 mL) and subjected to preparative reverse-phase HPLC. Intermediate 12 was obtained as white powder after lyophilization (3 mg, 55% yield).

Procedure for B1_Fmoc_Insulin:
Insulin (100 mg, 19.6 µmol) was dissolved in 5 mL phosphate buffer solution (pH = 6.2), Fmoc N-hydroxy-succinimide ester dissolved in dioxane (0.05 M, 1.5 equiv, 294 µL) was added into the insulin solution. The mixture was kept stirring for 30 minutes. The reaction solution was diluted with distilled deionized water (5 mL) and subjected to preparative reverse-phase HPLC, which gave the corresponding B1_Fmoc_insulin (10 mg).

Procedure for A1_benzyl Insulin (15):
B1_Fmoc_Insulin (10 mg, 1.66 µmmol), NaBH 3 CN (5 equiv, 8.29 µmmol) was dissolved in 600 µL citric acid buffer (pH = 6.1). 0.5 M benzaldehyde/DMSO solution (2 equiv, 6.62 µL) was added into the system and incubated at 37 o C for 24 hours. The reaction solution was diluted with distilled deionized water (3 mL) and subjected to preparative reverse-phase HPLC, which gave the corresponding modified product. Then it was dissolved in 20% piperidine/DMF and stirred for 20 minutes. The reaction solution was diluted with distilled deionized water (3 mL) and subjected to preparative reverse-phase HPLC, which gave the corresponding A1_benzyl insulin 15 (3.1 mg, 32% yield for two steps).

Procedure for A1_benzoyl Insulin (16):
B1_Fmoc_Insulin (10 mg, 1.66 µmmol) was dissolved in 800 µL phosphate buffer solution (pH = 6.2). 0.05 M benzoic anhydride/dioxane solution (2 equiv, 66.4 µL) was added into the system and incubated at 37 o C for 24 hours. The reaction solution was diluted with distilled deionized water (3 mL) and subjected to preparative reverse-phase HPLC, which gave the corresponding bezoylated product. Then it was dissolved in 20% piperidine/DMF and stirred for 20 minutes. The reaction solution was diluted with distilled deionized water (3 mL) and subjected to preparative reverse-phase HPLC, which gave the corresponding A1_benzoyl insulin 14 (0.9 mg, 9% yield).

Biological assays Insulin bioactivity assay
To determine the extent of insulin signaling induced by insulin molecules, pAkt Ser473 levels were measured in HEK293 cells, overexpressed with human IR-B. The cell line was cultured in DMEM with 10% FBS, pen/strep and 2 ug/mL puromycin. For the assay, 40,000 cells per well were plated in a 96-well plates with culture media containing 1% FBS. 24 hours later, 50 uL of insulin solution was pipetted into each well after the removal of the original media. After a 30-min treatment, the insulin solution was aspirated and the HTRF pAkt Ser473 kit (Cisbio, Massachusetts, USA) was used to measure the intracellular level of pAkt Ser473. Briefly, the cells were first treated with cell lysis buffer (50 uL per well) for 1 hour under mild shaking. 16uL of cell lysate was then added to 4uL of detecting reagent in a white 384well plate. After 4-hour incubation, the plate was read in a Synergy Neo plate reader (Biotek, Vermont, USA). The data was processed according to the manufacturer's protocol.

Fluorescein-labeled insulin imaging assay
A competitive binding imaging assay was used to determine the uptake of fluorescein-labeled insulin in the presence and absence of human insulin. First, a mouse fibroblast cell line, NIH 3T3, overexpressed with human IR-B was cultured in DMEM with 10% FBS, pen/strep and 2 ug/mL puromycin. For the assay, 20,000 cells per well were plated in a 96-well plates with culture media containing 1% FBS. 24 hours later, the media was aspirated and 1 µg/mL of fluorescein-labeled insulin was added to 16 wells and 10 µg/mL of human insulin was added to 8 wells containing 1 µg/mL fluorescein-insulin. These insulins were dissolved in an imaging buffer consisting of 140 mM NaCl, 2.5 mM KCl, 1.8 mM CaCl 2 , 1 mM MgCl 2 , 20 mM Hepes (mOsm = 300) pH 7.4 (Life Technologies Corporation). Immediately after addition of insulin solution, cells were imaged for a period of 30 minutes using a GFP filter and 10X magnification on an Axio Observer.A1.

Copy of LC chromatogram, MS spectrum, MS/MS spectrum, and trypsin digested segments MS spectrum.
TIC for 3a: MS Spectrum for 3a: TIC for A1_Benzyl_Insulin (15)