Rapid biocompatible macrocyclization of peptides with decafluoro-diphenylsulfone

Abstract

In this manuscript, we describe modification of Cys-residues in peptides and proteins in aqueous solvents via aromatic nucleophilic substitution (S_NAr) with perfluoroarenes (fAr). Biocompatibility of this reaction makes it attractive for derivatization of proteins and peptide libraries comprised of 20 natural amino acids. Measurement of the reaction rates for fAr derivatives by ¹⁹F NMR with a model thiol donor (β-mercaptoethanol) in aqueous buffers identified decafluoro-diphenylsulfone (DFS) as the most reactive S_NAr electrophile. Reaction of DFS with thiol nucleophiles is >100 000 faster than analogous reaction of perfluorobenzene; this increase in reactivity enables application of DFS at low concentrations in aqueous solutions compatible with biomolecules and protein complexes irreversibly degraded by organic solvents (e.g., bacteriophages). DFS forms macrocycles when reacted with peptides of the general structure X _n -Cys-X _m -Cys-X _l , where X is any amino acid and m = 1-15. It formed cyclic peptides with 6 peptide hormones-oxytocin, urotensin II, salmon calcitonin, melanin-concentrating hormone, somatostatin-14, and atrial natriuretic factor (1-28) as well as peptides displayed on M13 phage. Rates up to 180 M^-1 s^-1 make this reaction one of the fastest Cys-modifications to-date. Long-term stability of macrocycles derived from DFS and their stability toward oxidation further supports DFS as a promising method for modification of peptide-based ligands, cyclization of genetically-encoded peptide libraries, and discovery of bioactive macrocyclic peptides.

Fig. 1. (A) Design of reagents for perfluoroarene macrocyclization by cross-linking two Cys-residues in a protein in aqueous conditions. (B) Mechanism of S_NAr reaction. (C) Rate constants (k) of the reaction between substituted fAr and βME, as measured by ¹⁹F NMR, increase with the increasing electronegativity of R group. As a measure of electronegativity, we used pK_a in a series of phenylketones for which many values are known or can be calculated (ESI Fig. S4†). (D) To find the S_NAr reagents for rapid modification in water, we plotted molar concentration of fAr necessary to reach 50% conversion in 30 minutes (calculated as ln(2)/(1800 × k), where k is the rate constant) and % of organic co-solvent necessary to dissolve the fAr at this concentration. Most fAr are poorly reactive and/or too insoluble in aqueous solvents; only DFS and perfluoropyridine (fPy) can be used in conditions that require low amount of organic co-solvent.

Fig. 2. (A) S_NAr reaction of DFS with peptides is sequence and solvent-dependent. (B) Rates in all conditions were measured by monitoring absorbance at 320 nm, and identities of the products were confirmed by NMR and Liquid Chromatography Mass-Spectrometry (LCMS, ESI Fig. S7 and S12–S15†). (C) The rate of the reaction correlates with acidity of the thiol; pK_a were estimated by measuring the absorbance of peptides at 240 nm as described in ESI Fig. S18 and were in accordance with previously reported values for CXC peptides.

Fig. 3. (A and B) Cyclization of reduced oxytocin (OT) by DFS. Quenching the reaction by 2% TFA at 30 s revealed ∼90% consumption of starting material, 80% of product, and 12% of open-chain intermediates M1 and M2. (C) Stacked bar representation of the composition of reaction mixture determined by HPLC shows that both M1 and M2 of OT were completely converted to product by 5 min. Analogous studies with five other peptide hormones in the presence of 1 mM DFS. Fraction of the reactant, sum of M1 + M2 intermediates, and products was determined by integration of the HPLC traces at 215 nm (see ESI Fig. S16, S22, and S23 for raw traces).

Fig. 4. (A) Quantification strategy for the reaction between DFS and M13 phage displaying a peptide ACPARSPLEC. (B) Reaction of reduced phage with biotin-iodoacetamide (BIA) biotinylates the phage. (C) Capture assay measures the yield of biotinylation. (D) Capture assay determined that 95% of phage is reduced (reactive with BIA). Diluting the reducing agent 10-fold does not change the number of reduced thiols, but exposing the reduced phage to 0.5 mM DFS in 30% MeCN/Tris buffer for 30 min consumes reactive thiols yielding 80% OFS-modified phage. Error bars represent standard deviations from two experiments. (E) Capture can be summarized as stack bar: bottom bar shows that ∼60% of phage is modified by 0.5 mM solution of DFS in aqueous Tris buffer containing 5% DMF as co-solvent.

Fig. 5. (A) An aqueous solution of DFS containing 5% DMF as co-solvent can conveniently convert commercially available phage displayed library of 10⁹ disulfide heptapeptides—Ph.D. C7C from New England Biolabs—to a library of OFS-macrocyclic peptides. (B) Biotin capture, analogous to that described in Fig. 4C–E, quantified the efficiency of this modification. After 30 minutes to 2 hours reaction with DFS, 35–45% of the library is modified with DFS yielding ∼350–450 million of diverse phage-displayed OFS-macrocycles. Data is averaged from two independent experiments.

Fig. 6. Robust oxidation of SWCDYRC-OFS conjugate to peptide-aldehyde and subsequent one-pot conversion to a cyclic glycopeptide.