Native Chemical Ligation via N-Acylurea Thioester Surrogates Obtained by Fmoc Solid-Phase Peptide Synthesis

Abstract

Native chemical ligation (NCL) enables the direct chemical synthesis and semisynthesis of proteins of different sizes and compositions, streamlining the access to proteins containing posttranslational modifications (PTMs). NCL assembles peptide fragments through the chemoselective reaction of a C-terminal α-thioester peptide, prepared either by chemical synthesis or via intein-splicing technology, and a recombinant or synthetic peptide containing an N-terminal Cys. Whereas the generation of C-terminal α-thioester proteins can be achieved via the recombinant fusion of the sequence of interest to an intein domain, chemical methods can also be used for synthetically accessible proteins. The use of Fmoc -solid-phase peptide synthesis (Fmoc-SPPS) to obtain α-thioester peptides requires the development of novel strategies to overcome the lability of the thioester bond toward piperidine Fmoc-removal conditions. These new synthetic methods enable the easy introduction of PTMs in the thioester fragment. In this chapter, we describe an approach for the synthesis and use of C-terminal α-N-acylbenzimidazolinone (Nbz) and α-N-acyl-N′'-methylbenzimidazolinone (MeNbz) peptides in NCL. Following stepwise peptide elongation, acylation with p-nitrophenylchloroformate and cyclization affords the Nbz/MeNbz peptides. The optimization of the coupling conditions allows the chemoselective incorporation of the C-terminal amino acid (aa) on the 3,4diaminobenzoyl (Dbz) and prevents undesired diacylations of the resulting oaminoanilide. Following synthesis, these Nbz/MeNbz peptides undergo NCL straightforwardly at neutral pH catalyzed by the presence of arylthiols. Herein, we apply the Nbz technology solid phase synthesis, NCL-mediated cyclization and folding of the heterodimeric RTD-1 defensin, an antimicrobial peptide isolated from the rhesus macaque leukocytes.

On the Cyclization of Non‐cyclic Peptides for Biological Applications: Inspiration from Naturally Cyclic Peptides

“…Intramolecular native chemical ligation (NCL) is the most widely used technique for cyclizing linear cyclotide precursors containing a cysteine and an α-thioester group in the N-terminal and C-terminal ends respectively. [28] The process can be performed at physiological pH and can theoretically be accomplished between six cysteine amino acids found in the peptide structure. Yet, research on natural cyclotides (kalata B1 and MCoTIÀ I/II) suggested that the cyclization yield is highly improved when the cysteines are located in loops 3 and 6.…”

On the Utility of Chemical Strategies to Improve Peptide Gut Stability

“…The linear sequences were cyclized using an intramolecular version of NCL 157 via N- acylurea (Nbz) thioester surrogates. 120 , 158 , 159 Upon cyclization, the cyclotides were folded under oxidative conditions. 120 − 122 …”

“…Peptides were manually assembled on a H 2 N-Rink-ChemMatrix resin (1.0 mmol scale, 7-fold excess of amino acid, HBTU:DIPEA 7:10.5-fold activation, 30 min coupling time). The linear sequences were cyclized using an intramolecular version of NCL via N- acylurea (Nbz) thioester surrogates. ,, Upon cyclization, the cyclotides were folded under oxidative conditions. − …”

One‐Pot Chemical Protein Synthesis Utilizing Fmoc‐Masked Selenazolidine to Address the Redox Functionality of Human Selenoprotein F**

“…The corresponding C‐terminal peptides SELENOF(141‐165)(A141U) was synthesized by Fmoc‐SPPS, purified and characterized by HPLC and ESI‐MS (Scheme S1, Figure S1). Both SELENOF(96‐140)(Sec96Sez)‐COSR and SELENOF(89‐95)‐COSR (Schemes S2 and S3), bearing a C‐terminal thioester [27] were prepared similarly by standard stepwise Fmoc‐SPPS (further details for the syntheses can be found in Supporting Information 4.3 and Figures S2–S4) [27a,28] . The ligation between SELENOF(96‐140)(Sec96Sez)‐COSR and SELENOF(141‐165)(A141U) was performed at 37 °C for 18 h in the presence of TCEP and sodium ascorbate [29] to yield SELENOF(96‐165)(Sec96Sez/A141U) (Figure S5).…”

“…Both SELENOF(96‐140)(Sec96Sez)‐COSR and SELENOF(89‐95)‐COSR (Schemes S2 and S3), bearing a C‐terminal thioester [27] were prepared similarly by standard stepwise Fmoc‐SPPS (further details for the syntheses can be found in Supporting Information 4.3 and Figures S2–S4). [ 27a , 28 ] The ligation between SELENOF(96‐140)(Sec96Sez)‐COSR and SELENOF(141‐165)(A141U) was performed at 37 °C for 18 h in the presence of TCEP and sodium ascorbate [29] to yield SELENOF(96‐165)(Sec96Sez/A141U) (Figure S5). To convert Sec141 to Ala, the purified ligated product was subjected to deselenization reaction with TCEP at pH∼5, and under anaerobic conditions.…”