Stepwise triple-click functionalization of synthetic peptides

A sequence of click reactions and selective deprotection steps enables the stepwise synthesis of defined triple-functionalized peptides.


General information
The chemicals were obtained from Sigma Aldrich, Alfa Aesar, Acros Organics, ABCR, Flouorochem, Iris Biochem, Carbosynth or VWR and were used without further purification. Reactions with air-and moisture-sensitive reactants were performed under argon atmosphere and in anhydrous solvents.
Solutions were concentrated on a rotary evaporator from Heidolph equipped with a PC3001 VARIOpro pump from Vacuubrand. Column chromatography was carried out on silica gel 60Å (particle size: 40-60 μm) from Acros Organics. Solvents in the p.a. quality from Lach-Ner and Penta were used for elution. Mixtures of solvents are each stated as volume fractions. For flash column chromatography a CombiFlash ® Rf+ from Teledyne ISCO was used. Thin-layer chromatography was performed on aluminum sheets from Merck (silica gel 60 F254, 20 × 20 cm). Chromatograms were visualized by UV light (λ = 254 nm/ 366 nm) or by staining with KMnO 4 solution, (NH 4 ) 2 Ce(NO 3 ) 6 /(NH 4 ) 6 Mo 7 O 24 *4H 2 O solution, or PPh 3 /ninhydrin method. For amino acid analysis or determination of resin loading, samples were hydrolyzed in 6 M HCl at 110~C overnight and analyzed on a Biochrom 30 amino acid analyzer (Biochrom Ltd., UK). 1 H-and 13 C-NMR spectra were measured on a Bruker Avance III™ HD 400 MHz NMR system equipped with Prodigy cryo-probe or on a Bruker Avance III™ HD 500 MHz Cryo. CDCl 3 , methanol-d 4 deuterium oxide and DMSO-d 6 from Sigma Aldrich or Eurisotop were used as solvents. Chemical shifts δ are quoted in ppm in relation to the chemical shift of the residual non-deuterated solvent peak (CDCl 3  . J values are given in Hz. High-resolution mass spectra were recorded on an Agilent 5975C MSD Quadrupol, Q-Tof micro from Waters or LTQ Orbitrap XL from Thermo Fisher Scientific. HPLC-MS measurements were performed either on an LCMS-2020 system from Shimadzu equipped with Luna® C18(2) column (3µm, 100A, 100 × 4.6 mm), or on HPLC-MS Infinity 1260 system equipped with 6120 Quadrupole LC/MS detector from Agilent Technologies and either preparative column Luna® 5 m C18 (2), 100 Å, 250 x 21.2 mm (Phenomenex) or analytical column Poroshell 120, EC-C18 4 m, 4.6 x 100 mm (Agilent Technologies). UV/VIS spectroscopy was performed on a Cary 60 UV/Vis spectrophotometer from Agilent Technologies. Data from experiments were processed using Microsoft Excell 2016 MSOs software. Fluorescence measurements were performed on Spark® microplate reader from Tecan, in 96-well half area black polystyrene microplates (Corning). Automated peptide synthesis was done on PS3™ Peptide Synthesizer, Protein Technologies, Inc.. For microwave irradiation, the standard kitchen microwave oven Daewoo KOR-9GPBC (Daewoo Electronics) was used.

Synthetic procedures
Compounds prepared according to literature: AA building blocks synthesis: General procedure 1 (for synthesis of 5 and 6): According to lit. 3 , with slight modifications. Alkyne was taken into dry THF and cooled to -78 °C in acetone/dry ice bath. The solution of n-BuLi (2.5M in hexane; 1.3 eq.) was added slowly and reaction mixture was stirred for 10 min. The cooling bath was replaced with ice bath (0 °C) and Silyl-Cl (1.2 eq.) was added dropwise. The reaction proceeded for 3 hrs at r.t., was quenched with sat. aq. NH 4 Cl, THF was gently evaporated, resulting slurry diluted with water, extracted with EtOAc, and concentrated. The crude product was purified by column chromatography.

tert-Butyl 5-(triethylsilyl)pent-4-ynoate (6)
The general procedure 1 was followed, using 43 mmol of 4. The reaction was quenched with sat. aq. NH 4 Cl (160 ml), THF was removed, resulting slurry diluted with water (200 ml), extracted with EtOAc (3 x 100 ml) and purified by silicagel column using PE-Et 2 O (50 : 1). Yield: 3.3 g, 29 %. NMR data correspond to lit. 3  General procedure 2 (for synthesis of 9, 10, and 11) According to lit. 6 , with slight modifications. To the solution of acid and N-hydroxy succinimide (1.05 eq.) in dry THF at 0 °C DCC (1.05 eq.) dissolved in dry THF was added slowly. The mixture was stirred for 40 min. at 0 °C, warmed gradually to r.t. and stirred for additional 2h. The reaction mixture was filtered, concentrated, dissolved in EtOAc, filtered, washed with saturated solution of NaHCO 3 and brine, and dried over Na 2 SO 4 . The residue was swiftly purified on column of silica.

General procedure 3 (for synthesis of 1, 2 and 3)
A suspension of Fmoc-L-Lys-OH in the mixture of dioxane and sat. aq. NaHCO 3 (1 : 3) was cooled to 0 °C. A solution of active ester (1.2 eq.) in dioxane was added dropwise. In 5 min the ice bath was removed and reaction proceeded at r.t. for 20 hrs. Reaction mixture was diluted with EtOAc, washed with sat. aq. citric acid, water, brine and dried over Na 2 SO 4 . Concentrated residue was purified by flash chromatography on C18 phase and lyophilized. The general procedure 3 was followed, using 0.54 mmol of Fmoc-L-Lys-OH in the mixture of dioxane and sat. aq. NaHCO 3 (1 : 3; 4 ml) and 11 in dioxane (2 ml). For the workup, the reaction mixture was diluted with EtOAc (30 ml), washed with sat. aq. citric acid (20 ml), water (20 ml), brine (20 ml) and dried over Na 2   The general procedure 3 was followed, using 1.81 mmol of Fmoc-L-Lys-OH in the mixture of dioxane and sat. aq. NaHCO 3 (1 : 3, 12 ml) and 9 in dioxane (6 ml). For the workup, the reaction mixture was diluted with EtOAc (100 ml), washed with sat. aq. citric acid (25 ml), water (25 ml), brine (25 ml) and dried over Na 2
The general procedure 5 was followed using 1.48 mmol of 13, 300 mg of Dowex  1x8 and 10 ml of dry MeOH. The crude product was purified by flash chromatography in DCM -MeOH (520 %) and lyophilized. Yield: 382, 93 %.

Prepration of model peptides Pep14 and Pep15:
Resin loading: TentaGel-S-OH resin (269 mg, theor. 0.065 mmol) was soaked in dry DMF (2 ml) for 1.5 h and drained. In separate flask, Fmoc-Gly-OH (594.6 mg; 2 mmol) was taken into dry DMF (1.5 ml), dry DCM (6 ml) was added, the solution was cooled to 0 ºC, DIC (155 l; 1 mmol) was added. The reaction mixture was stirred at 0 ºC for 45 min. DCM was removed, the residue was dissolved in dry DMF, and the resulting solution was added to the drained resin. DMAP (2.44 mg; 0.02 mmol) was added, the syringe was flushed with argon and rotated on rotary shaker for 2 hrs. Resin was drained, washed with DMF (5x), DCM (5x) and dried. Yield: 257 mg of dried loaded resin. Loading by Fmoc estimation: 0.188 mmol/g; AA analysis: 0.137 mmol/g.

Fmoc estimation method:
Done according lit. 14 Dry loaded resin (approx. 5 mg, weighted precisely) was treated by DBU (2 % in DMF; 2 ml) for 40 min. The solution was collected, diluted to 10 ml with ACN, resulting solution was diluted for the second time (0.8 ml of solution to 10 ml by ACN), and its' optical density measured on UV spectrometer in optical cell (1 cm The following Fmoc protected amino acids were utilized: Step 1: Fmoc-L-Lys(pentynoyl-TES)-OH (2) for Pep14, Fmoc-L-Lys(pentynoyl-TIPS)-OH (3) for Pep15 Step 2: Fmoc-L-Tyr(tBu)-OH Step 3: Fmoc-L-Val-OH Step 4: Fmoc-L-Lys(Boc)-OH Step 5: Deprotection -removal of Fmoc group The resin was transferred from synthesizer reaction vessel to syringe (10 ml) equipped with sintered filter, washed with DCM (6x), drained, and dried. Yield: 143 mg (Pep14), resp. 166 mg (Pep15) of loaded resin. Small sample of resin (approx. 2 mg) was treated with NaOH following the general procedure bellow and the progress of the synthesis was verified by LC-MS analysis.

SPPS
Solid phase peptide synthesis was performed "by hand" following the lit. 15 .
The resin was mixed with N 2 stream for 30 min. The resin was drained; analytical sample was washed coupling conversion verified by ninhydrin test.

Synthesis of model modified peptide Pep1
Peptide Pep1 was synthesized on PS3 peptide synthesizer using L-glycine-loaded resin, standard Fmoc chemistry and 4 (resp. 2) equivalents of animo acid for coupling step. For detailed procedure see synthesis of Pep14.

Automated SPPS
Automated peptide synthesis was performed on PS3 peptide synthesizer, Protein Technologies, Inc. Synthesizer was loaded with methionine substituted resin (250 mg; 0.049 mmol). Resin was capped with acetic anhydride under standard automated protocol, than peptide was synthesized under standard automated Fmoc protocols, using either 4 equivalents of each commercial amino acid and 4 equivalents of HBTU as coupling agent, or 2 equivalents of modified amino acid 2 or 3 and 2 equivalents of HBTU. The remaining Fmoc-group was removed after the last coupling step by piperidine : DMF (1 : 4).

Standard protocols for SP3 Peptide Synthesizer:
For deprotection and coupling protocol details see synthesis of Pep14. Mixing of capping solution in vial 30 s 1 3 Capping 20 min.

Automated SPPS
Automated peptide synthesis was performed on PS3 peptide synthesizer, Protein Technologies, Inc.. Synthesizer was loaded with glycine-substituted resin (275 mg; 0.046 mmol). Resin was capped with acetic anhydride under standard automated protocol, than peptide was synthesized under standard automated Fmoc protocols, using either 4 equivalents of each commercial amino acid and 4 equivalents of HBTU as coupling agent, or 2 equivalents of modified amino acid 1 or 2 and 2 equivalents of HBTU. The remaining Fmoc-group was removed after the last coupling step, the deprotection solution was piperidine : DMF (1 : 4). For the standard protocol details see synthesis of Pep14 (coupling and deprotection and capping step) The following Fmoc protected amino acids were utilized: Step 1: Fmoc-L-Met-OH Step 2: Fmoc-Gly-OH Step 3: Fmoc-L-Lys(pentynoyl)-OH (1) Step 4: Fmoc-L-Phe-OH, Step  Optimization of TES removal from resin bound peptides: As AgClO 4 showed up to be the most convenient cleaving agent for removal of TES in the presence of TIPS, we used it to establish the procedure for semiorthogonal deprotection of resin-bound glycopeptides. We prepared the model resin-bound alkyne-modified peptides Pep14 (TES), Pep15 (TIPS), and Pep16 (TES + TIPS) and exposed them to AgClO 4 solutions of varying concentrations. Deprotected peptides were cleaved-off from the resin and analyzed by LC-MS (Shimadzu).
neutralized by HCl (0.2M in H 2 O) and the liquids were collected. Drained resin was washed with warm ACN (2x), ACN washes were mixed with previously collected liquids and analyzed by LC-MS.

Optimization of CuAAC reaction for resin-bound peptides:
In order to find the optimal conditions to modify peptides by CuAAC on solid phase, model resinbound heptapeptide Pep1 was "clicked" with saccharide analog Gal-C3-N 3 . In our hands, the first modification of peptide backbone was the most challenging one. LC-MS was used to analyze reaction mixture after product was cleaved-off from the resin, conversion was determined from peak areas of product Pep17 and remaining unmodified Pep1.

Fluorescence assay -Cleavage of fluorogenic substrate Pep11 by trypsin
Peptide Pep11 was subjected to the brief "proof-of-concept" assay with trypsin. Enzyme trypsin is able to cleave peptide chains after polar amino acid residues (arginine and lysine). The peptide Pep11, contains the fluorophore and the quencher molecule in close proximity so that the fluorescence of the coumarin is quenched. Cleavage of the peptide Pep11 by trypsin after the remaining lysine residue yields two short peptide sequences where the two molecules (the dye and the quencher) are apart. As consequence the fluorescence of the free coumarin can be observed and detected.