A versatile o-aminoanilide linker for native chemical ligation

Abstract

Chemical protein synthesis (CPS) is a consolidated field founded on the high chemospecificity of amide-forming reactions, most notably the native chemical ligation (NCL), but also on new technologies such as the Ser/Thr ligation of C-terminal salicylaldehyde esters and the α-ketoacid-hydroxylamine (KAHA) condensation. NCL was conceptually devised for the ligation of peptides having a C-terminal thioester and an N-terminal cysteine. The synthesis of C-terminal peptide thioesters has attracted a lot of interest, resulting in the invention of a wide diversity of different methods for their preparation. The N-acylurea (Nbz) approach relies on the use of the 3,4-diaminobenzoic (Dbz-COOH) and the 3-amino-(4-methylamino)benzoic (MeDbz-COOH) acids; the latter disclosed to eliminate the formation of branching peptides. Dbz-COOH has been also used for the development of the benzotriazole (Bt)-mediated NCL, in which the peptide-Dbz-CONH₂ precursor is oxidized to a highly acylating peptide-Bt-CONH₂ species. Here, we have brought together the Nbz and Bt approaches in a versatile linker, the 1,2-diaminobenzene (Dbz). The Dbz combines the robustness of MeDbz-COOH and the flexibility of Dbz-COOH: it can be converted into the Nbz or Bt C-terminal peptides. Both are ligated in high yields, and the reaction intermediates can be conveniently characterized. Our results show that the Bt precursors have faster NCL kinetics that is reflected by a rapid transthioesterification (<5 min). Taking advantage of this major acylating capacity, peptide-Bt can be transselenoesterified in the presence of selenols to afford peptide selenoesters which hold enormous potential in NCL.

Scheme 1. Previous work: synthetic avenues of 1,2-diaminobenzoyl derivatives for chemical protein synthesis. Following the route A, the peptide1–Dbz/MeDbz–CONH–resin is converted in the peptide1–Nbz/MeNbz–CONH₂ on resin or in solution through a phosgenation-type process. Next, this peptide is ligated through NCL to another Cys-peptide2. In the route B, the peptide1–Dbz–CONH₂ is transformed in peptide1–Bt–CONH₂ and then ligated to a Cys-peptide2. P = protecting group.

Scheme 2. This work: (A) the 1,2-diaminobenzene–PAL strategy developed in this work for the synthesis of o-aminoanilide–PAL peptides. (B) Route I: synthesis of the peptideXaa₁–(pCN-Phoc)Dbz and NCL. Route II: preparation of the peptideXaa₁–Dbz precursor of the peptideXaa₁–Bt and NCL mediated by peptideXaa₁–(4-MPOH) thioesters (II1) and peptideXaa₁–COSePh selenoesters (II2).

Fig. 1. (A) Scheme of the NCL between LYRAA–(pCN-Phoc)Dbz (12, 2 mM) and CRAFS (13, 3.0 mM) in 6 M Gdm·HCl, 0.2 M NaPhos, 0.2 M 4-MPOH, and 50 mM TCEP·HCl at pH 7.0. (B) HPLC traces at 220 nm of the ligation at different times. ® = l-Tyr, # = 2-benzimidazolinone (Nbz); ϕ = p-cyanophenol. (C) Chemical structures of the intermediates LYRAA–(pOH-phenylthiocarbonyl)Dbz (15), LYRAA–Nbz (16) and LYRAA–(4-MPOH) (17).

Fig. 2. (A) Kinetics of the ligation between CLAFS and LYRAG–(pCN-Phoc)Dbz–CONH–G (18) and LYRAG–(pCN-Phoc)MeDbz–CONH–G (19) and LYRAG–(pCN-Phoc)Dbz (20). (B) Plot of the fraction ligated of LYRAXaa₁CXaa₂AFS. (C) Formation of LYRAVCTAFS and LYRIPCTAFS. (D) Plausible mechanism for the ligation. Reactions were carried out at pH 6.8–7.0 in 6 M Gdm·HCl and 0.2 M NaPhos at 22 °C. Concentrations of the peptides were: [LYRAXaa₁–(pCN-Phoc)Dbz, LYRIP–(pCN-Phoc)Dbz] = 2 mM; [CXaa₂AFS] = 3–3.5 mM.

Fig. 3. (A) Scheme of the stepwise ligation between LYRAA–Dbz (25, 2.5 mM) and CTAFS (27, 4.7 mM): (i) NaNO₂ (3.5 eq.) oxidation at −15 °C, pH = 3, and 30 min; (ii) addition of 4-MPOH (200 mM) and TCEP·HCl (50 mM) at pH = 6.7; (iii) NCL, addition of CTAFS (27). (B) HPLC traces at 220 nm of the reaction after (i) (NaNO₂ addition), (ii) (4-MPOH addition) and (iii) (CTAFS addition) steps. -- = 4-MPOH.

Fig. 4. (A) Scheme of the stepwise ligation between LYRAF–Dbz (29, 2.2 mM) and CTAFS (27, 4.7 mM): (i) NaNO₂ (3.5 eq.) oxidation at −15 °C; pH = 3, 30 min; (ii) addition of (PhSe)₂ (50 mM) and TCEP·HCl (100 mM) at pH = 6.2. (B) HPLC traces at 220 nm of the reaction after addition of (PhSe)₂ and TCEP·HCl at 15 s and 15 min.

Fig. 5. (A) X-Ray structure of the homodimer DNA binding domain (DBD) of the glucocorticoid receptor (GR) bound to the GRE site (AGAACAaaaTGTTCT). The red color denotes the α-helix, green the β-strand, orange the interconnecting loops and blue the Zn atoms. (B) Sequence of the Zn finger domain of the human GR and fragments F1, F2 and F3 used for CPS. (C) Synthetic scheme of the GR assembled linearly from three fragments: first F2 + F3; second F1 + F2–F3.

Fig. 6. (A) HPLC traces at 220 nm of the CPS of the GR DBD: (i) F2(Acm)–MESH + F3 at 2 min and (ii) at 4 h, (iii) Acm removal from F2(Acm)–F3, (iv) F1-(pCN-Phoc)Dbz + F2–F3 at 2 min, (v) 2 h and (vi) 6 h, (vii) analytical HPLC trace at 220 nm of a purified sample of F1–F2–F3. ACN = acetonitrile. ϕ = p-cyanophenol. (B) ESIMS of F1–F2–F3 (Lys⁴¹⁹–Lys⁴⁹⁵) and the deconvoluted mass spectrum. (C) Circular dichroism spectra of the GR DBD (35 μM) in the presence of increasing concentrations of ZnCl₂: 0, 5, 10, 20, and 40 μM. (D) Electrophoretic mobility shift assay of the GR DBD and GRE. [GR DBD] lanes 1–7 = 0, 0.6, 0.8, 1.0, 1.4, 1.8, and 2.3 μM.