Head‐to‐tail cyclization of side chain‐protected linear peptides to recapitulate genetically‐encoded cyclized peptides

Abstract Genetically‐encoded cyclic peptide libraries allow rapid in vivo screens for inhibitors of any target protein of interest. In particular, the Split Intein Circular Ligation of Protein and Peptides (SICLOPPS) system exploits spontaneous protein splicing of inteins to produce intracellular cyclic peptides. A previous SICLOPPS screen against Aurora B kinase, which plays a critical role during chromosome segregation, identified several candidate inhibitors that we sought to recapitulate by chemical synthesis. We describe the syntheses of cyclic peptide hits and analogs via solution‐phase macrocyclization of side chain‐protected linear peptides obtained from standard solid‐phase peptide synthesis. Cyclic peptide targets, including cyclo‐[CTWAR], were designed to match both the variable portions and conserved cysteine residue of their genetically‐encoded counterparts. Synthetic products were characterized by tandem high‐resolution mass spectrometry to analyze a combination of exact mass, isotopic pattern, and collisional dissociation‐induced fragmentation pattern. The latter analyses facilitated the distinction between targets and oligomeric side products, and served to confirm peptidic sequences in a manner that can be readily extended to analyses of complex biological samples. This alternative chemical synthesis approach for cyclic peptides allows cost‐effective validation and facile chemical elaboration of hit candidates from SICLOPPS screens.


Manual peptide synthesis
During manual peptide elongation, the highly-reactive HATU amide bond coupling reagent was chosen to reduce reaction times and avoid resort to repeated couplings. To facilitate the optimal use of HATU under anhydrous conditions, resin for manual SPPS was loaded into disposable polypropylene syringes fitted with polypropylene frits (70 µm porosity) and needles rather than filter tubes. 1 To remove adventitious water from resin-charged fritted syringes, dry solvents (DMF or CH2Cl2) under nitrogen were repeatedly (2)(3)(4)(5) times) aspirated into and expelled from the syringe over 5-30 minutes.
Manual peptide elongation procedure 2-ClTrt resin bearing an N-terminal Fmoc amino acid residue (0.25 mmol nominal loading) was deprotected by treatment with two batches of piperidine (20%) in DMF (3-4 mL) over a total of 17 min (2 min, then 15 min) and the liquid phase was removed. The solid phase was washed multiple times with DMF (5x over 5 min) and CH2Cl2 (2x over 2 min) to afford a resin-bound free amine. The latter was elongated by treatment with a dry DMF (2.8-3 mL) solution of the subsequent Fmoc-amino acid (0.750 mmol, 3 equiv.), HATU (271 mg, 0.713 mmol, 2.85 equiv.), and Hünig's base (258 µL, 1.48 mmol, 5.9 equiv.). After mixing by repeated inversion at room temperature for 45 min, the reaction medium was removed and the resin was washed multiple times with DMF (5x over 5 min) and CH2Cl2 (2x over 2 min). To confirm reaction completion, resin aliquots from before and after the acylation reaction were subjected to the Kaiser colorimetric test and compared. 2 The deprotection/elongation sequence was appropriately iterated according to the targeted sequence to afford an Fmoc-peptidyl resin, which was deprotected by repeating the piperidine process to afford the final N-terminal free amine peptidyl resin 2. The latter was used directly in the TFE-mediated Trt resin cleavage procedure described below.
After mixing by argon bubbling for 10 min at room temperature (step 15), the liquid phase was removed and the resin was washed with DMF (10 mL, step 16). The capping sequence was repeated (steps 17-20) and the resin was washed with additional DMF (20 mL total, steps 21-22) to afford 2-ClTrt resin bearing a single Fmoc amino acid. The latter was used directly in subsequent elongation reactions by assuming quantitative yield (0.25 mmol) for subsequent stoichiometry calculations. Figure S1. Screenshot of the automated method used for loading Fmoc-amino acids onto 2-ClTrt-Cl resin using a Liberty1 instrument.
Automated peptide elongation procedure.
Resin-bound amino acid was achieved using the recommended precursors, reagents, and solvents, including DMF (peptide grade) solutions of Fmoc-amino acids (0.2 M), HBTU (0.5 M), Hünigs base (35%), and piperidine (20%). 3 The default microwave heating setting for the coupling was changed from 75 °C to 50 °C and the default 300 s coupling reaction time setting was changed to 1800 s. As final automated step, the resin was treated with piperidine (20%) in DMF, heated to 50 °C for 30 min, and washed with DMF to afford N-terminal free amine peptidyl resin 2. The latter was transferred from the synthesizer into a fritted syringe for direct use in the TFE-mediated resin cleavage procedure.

TFE-mediated Trt resin cleavage procedure
N-terminal free amine peptidyl resin 2 was treated with TFE (30%) in CH2Cl2 (12 mL), mixed by periodic inversion for 1 h at room temperature, and the liquid phase was collected. The cleavage was repeated using fresh TFE/ CH2Cl2 mixture and the combined liquid phases were concentrated in vacuo. The resulting crude side-chain protected peptide 3 was employed directly in the linear peptide macrocyclization procedure.

Peptide macrocyclization procedure
A solution of NEt3 (35 µL, 250 µmol, 10 equiv.) in CH3CN (11.2 mL) was added to a DMSO (470 µL) solution of linear peptide 3 (25 µmol, 1 equiv.) and the resulting mixture was treated with DEPBT (18.6 mg, 62 µmol, 2.5 equiv.). After stirring at room temperature for 24 h, the reaction was quenched with AcOH (0.5-1 mL) and the volume was reduced in vacuo by ≥ 10 fold (to 1-2 mL). The resulting DMSO/CH3CN solution of the crude was filtered (3 mm syringe filter, 0.2 µm pore size) and purified by preparatory HPLC to afford cyclic peptides 4, 5, or a 4/5 mixture dependent on sequence. Preparatory HPLC purifications were performed on an Agilent 1200 instrument or a Waters Inc. (Milford, MA) 2795 coupled to a 2996 diode array and micromass ZQ for UV and MS detection respectively. Cyclic peptides 4 were eluted using flow rates of 20 mL/min under the conditions detailed in Table S1. The collected fractions were concentrated in vacuo (2)(3)(4)(5) at 50 °C, then concentrated to dryness with assistance from multiple azeotropic coevaporations with i-PrOH or 1,4-dioxane as necessary. Table S1. Methods used for preparatory HPLC purification.

Gradient
Peak detection

TFA-mediated deprotection procedure
To avoid known complications associated with the deprotection of peptides containing Cys(Trt) and Trp(Boc) residues, the cleavage cocktail containing DTT as nucleophilic scavenger was employed. 6 Among alternative thiols, DTT was selected due to reduced stench.
Otherwise, purification was conducted with DMSO solutions using preparative HPLC. The collected fractions were concentrated in vacuo (1-5 Torr) at 50 °C, then concentrated to dryness with assistance from multiple azeotropic coevaporations with i-PrOH or 1,4-dioxane. The residue was dissolved in dilute HCl(aq) (> 10 equiv.), and lyophilized to yield 1, 6, or a 1/6 mixture, typically as HCl salts, dependent on structure.

Chromatographic characterization of peptidic products
The purity and identity of synthetic  Table S2.
Cyclic peptides 1 and 6 were analyzed using a Dionex/Thermo UltiMate 3000 binary RSLCnano Ultra High Performance Liquid Chromatography (UHPLC) system coupled to a Q-Exactive MS operating under the chromatographic and spectrometric conditions detailed in Table S2 and Table S3. Purified cyclic peptides 1 harbouring Cys residues were prone to dimerization by disulfide bond formation, complicating mass spectrometry analyses, which was avoided by adding TCEP (0.5 mM final concentration) reductant to samples prior to injection.

Synthesis of cyclic KCKPFKSI
Intermediate 3g