Synthesis of multi-module low density lipoprotein receptor class A domains with acid labile cyanopyridiniumylides (CyPY) as aspartic acid masking groups

Abstract

Low-density lipoprotein receptor class A domains (LA modules) are common ligand-binding domains of transmembrane receptors of approximately 40 amino acids that are involved in several cellular processes including endocytosis of extracellular targets. Due to their wide-ranging function and distribution among different transmembrane receptors, LA modules are of high interest for therapeutic applications. However, the efficient chemical synthesis of LA modules and derivatives is hindered by complications, many arising from the high abundance of aspartic acid and consequent aspartimide formation. Here, we report a robust, efficient and general applicable chemical synthesis route for accessing such LA modules, demonstrated by the synthesis and folding of the LA3 and LA4 modules of the low-density lipoprotein receptor, as well as a heterodimeric LA3-LA4 constructed by chemical ligation. The synthesis of the aspartic acid-rich LA domain peptides is made possible by the use of cyanopyridiniumylides (CyPY) - reported here for the first time - as a masking group for carboxylic acids. We show that cyanopyridiniumylide masked aspartic acid monomers are readily available building blocks for solid phase peptide synthesis and successfully suppress aspartimide formation. Unlike previously reported ylide-based carboxylic acid protecting groups, CyPY protected aspartic acids are converted to the free carboxylic acid by acidic hydrolysis and are compatible with all common residues and protecting groups. The chemical synthesis of Cys- and Asp-rich LA modules enables new access to a class of difficult to provide, but promising protein domains.

Fig. 1. (A) Low density lipoprotein receptor (LDLR) consists of a cytosolic domain, EGF-like domain, β-propeller domain and several LA modules (LA module reproduced from PDB 1AJJ, ref. 32). (B) Overview of LA modules that were chemically synthesized in this work. (C) Comparison of ylide masked aspartic acid building blocks with the typically used aspartic acid building block Fmoc-Asp(O^tBu)-OH.

Scheme 1. Aspartimide is formed by the base-catalyzed attack of the backbone amide onto the ester protected aspartic acid sidechain. Several by-products are formed upon ring-opening by nucleophiles such as water and piperidine.

Fig. 2. (A) Synthesis of cyanopyridiniumylides 1–5 from hydrocinnamic acid 6 and pyridinium salt 7–11 (7 R = H, 8 = 4-OCH3, 9 = C(O)NHCH3, 10 = 3-Cl, 11 = 4-Ph). The crystal structure of cyanopyridiniumylide 1 confirms Z configuration. (B) Postulated mechanism for the conversion of cyanopyridiniumylides to the free carboxylic acid using (i) acidic conditions (ii) electrophilic halogen species. (C) Reaction rates of hydrolysis of 1–4 under acidic conditions as determined by 1H-NMR. 4-Phenyl substituted cyanopyridiniumylide 5 was insoluble under the given conditions.

Fig. 3. (A) Synthesis of Fmoc-Asp(CyPY)-OH 12 from commercially available Fmoc-Asp(OH)-O^tBu 14 and pyridinium salt 7. (B) Model peptide 16 synthesized using conventional building block Fmoc-Asp(O^tBu)-OH and Fmoc-Asp(CyPY)-OH 12. 15-CyPY and 15-O^tBu were incubated with (a) 20 v% piperidine in DMF (16 h, rt) or (b) 2 v% DBU (1 h, rt) to evaluate their ability to prevent formation of aspartimide (15-Aspartimide). (C) Sequence of aspartimide prone NN92 17 containing an Asp-Gly motif.

Fig. 4. (A) Synthesis of monomeric LA modules R3 18 and R4 19. Aspartic acids residues able of aspartimide formation are highlighted by blue circles. Synthesis utilizing Fmoc-Asp(CyPY)-OH 12 and Fmoc-Asp(O^tBu)-OH allowed comparison of the two monomers. (B) Synthesis of dimeric LA module R3–R4 (Thr42Hse) 24 using KAHA ligation. Acm-deprotection conditions: AcOH/H₂O (1/1, v/v), 1 w% AgOAc, 45 °C, 2 h; Folding conditions: NaCl (150 mM), CaCl₂ (2.5 mM), TRIS (50 mM), GSH (3 mM), GSSG (0.3 mM), pH 8.5, 4 °C, 16 h.