Alternative chemistries for the synthesis of thrombospondin‐1 type 1 repeats

The synthesis of a novel protein target can be technically demanding due to the large number of peptide functional groups and numerous sequence-specific chemical and conformational challenges that may be encountered. The assembly of polypeptides by native chemical ligation (NCL) is typically robust when peptides are solubilized using one of a range of chaotropic agents, detergents, or organic cosolvents. In contrast, the synthesis of the peptide fragments can be subject to challenges inherent in solid-phase peptide synthesis (SPPS) as well as limitations specific to the generation of peptides containing C-terminal thioesters or N-terminal cysteines. Herein we describe a series of synthetic approaches for the total chemical synthesis of the second type 1 repeat of thrombospondin-1 (TSR2) that vary in both ligation site and α-amine protecting group strategy (Boc/Fmoc). The results demonstrate the chemistry and context-dependence of challenging sequences, as well as how such challenges can be overcome by altering the chemistry used for SPPS, the choice of ligation site, and the resin used as a solid support. The first synthetic route to the ~7 kD TSR2 domain involved a two-piece ligation of fragments synthesized using Boc/Bzl solid phase peptide synthesis (Boc-SPPS)1. However, despite attempts to improve synthetic yields by changing the ligation site and resins used for the synthesis, efforts to scale up to produce protein for biophysical analysis were hampered by low purification yields of peptide fragments. As a result, alternative synthetic routes to these ligation fragments were explored, including the use of Fmoc/tBu SPPS for both N- and C-terminal fragments. Compared to Boc-SPPS, Fmoc-based SPPS is more widely practiced, avoids the need for hydrofluoric acid (HF), and is more compatible with automation. The use of Fmoc-SPPS for the synthesis of peptide precursors for NCL has been encouraged by the development of new approaches for the synthesis of thioesters by Fmoc-SPPS2–5. We report the first application of the N-acylurea (Nbz) activation strategy to the synthesis of a protein domain. The second type 1 repeat from human thrombospondin-1 is a representative TSR domain with known structure6 and activity7. While the folded domain is structurally defined as residues Q416-I473, the leading KRFK sequence has been implicated in the activation of TGF-beta8 and adds positively charged residues that improve the solubility of unprotected peptide intermediates1. The synthetic target described in this work, mTSR2, comprises residues K412-I473 of human thrombospondin-1 with Met451Nle and Asn452Gly and amidated termini, as previously described1. A two-piece ligation strategy was designed to facilitate modifications to one half of the protein without requiring resynthesis of the entire protein. Inspection of the peptide sequence revealed that, of the six cysteines in the protein, three were centrally located and had good potential as ligation sites1. In order to determine which ligation site would be the most useful for the synthesis of multiple TSR analogs, three distinct pairs of N and C-terminal peptides were synthesized (Figure 1). Figure 1 Sequence of mTSR2 and fragments thereof, corresponding to the three potential ligation sites for TSR2 (A, B, and C). Z represents norleucine. X represents either a thioester (1a, 3a, 5a) or N-acyl-benzimidazole (Nbz) thioester precursor (1b, 3b, 5b). ... Results Fmoc-Based SPPS of Ligation Fragments Synthetic access to five of the six peptides, 2–6, corresponding to the three potential ligation sites A–C for TSR2 (Figure 1) was obtained. Synthesis of peptide 1a, the longest N-terminal fragment (ligation site A), encountered obstacles that were not easily overcome, leading us to focus on the two alternative ligation sites, B and C, presented in the molecule. The most robust synthetic access was established using N-and C-terminal fragments corresponding to ligation site B and a combination of Boc and Fmoc-SPPS.