A thiol group introduced on the γ-carbon of lysine mediates robust native chemical ligation at both the α- and ε-amines in two consecutive steps. Desulfurization then affords the final product, in which the lysine residue at the ligation site has an isopeptide bond on its side chain. The method is useful for the synthesis of proteins containing special post-translational modifications on lysine.

AbstractButelase-mediated ligation (BML) can be used to modify live bacterial cell surfaces with diverse cargo molecules. Surface-displayed butelase recognition motif NHV was first introduced at the C-terminal end of the anchoring protein OmpA on E. coli cells. This then served as a handle of BML for the functionalization of E. coli cell surfaces with fluorescein and biotin tags, a tumor-associated monoglycosylated peptide, and mCherry protein. The cell-surface ligation reaction was achieved at low concentrations of butelase and the labeling substrates. Furthermore, the fluorescein-labeled bacterial cells were used to show the interactions with cultured HeLa cells and with macrophages in live transgenic zebrafish, capturing the latter's powerful phagocytic effect in action. Together these results highlight the usefulness of butelase 1 in live bacterial cell surface engineering for novel applications.

AbstractButelase-mediated ligation (BML) can be used to modify live bacterial cell surfaces with diverse cargo molecules. Surface-displayed butelase recognition motif NHV was first introduced at the C-terminal end of the anchoring protein OmpA on E. coli cells. This then served as a handle of BML for the functionalization of E. coli cell surfaces with fluorescein and biotin tags, a tumor-associated monoglycosylated peptide, and mCherry protein. The cell-surface ligation reaction was achieved at low concentrations of butelase and the labeling substrates. Furthermore, the fluorescein-labeled bacterial cells were used to show the interactions with cultured HeLa cells and with macrophages in live transgenic zebrafish, capturing the latter's powerful phagocytic effect in action. Together these results highlight the usefulness of butelase 1 in live bacterial cell surface engineering for novel applications.

For the proteins that cannot be expressed exactly by cell expression technology (e.g., proteins with multiple posttranslational modifications or toxic proteins), chemical synthesis is an important substitute. Given the limited peptide length offered by solid-phase peptide synthesis invented by Professor Merrifield, peptide ligation plays a key role in long peptide or protein synthesis by ligating two small peptides to a long one. Moreover, high-molecular-weight proteins must be synthesized using two or more peptide ligation steps, and sequential peptide ligation is such an efficient way. In this paper, we reviewed the development of chemical protein synthesis, including solid-phase peptide synthesis, chemical ligation, and sequential chemical ligation.

Peptide Weinreb amide derivatives with an N-substituted mercaptoethyl group are designed as thioester precursors for native chemical ligation. We show that these amides undergo rapid ligation with a cysteinyl peptide under normal NCL conditions to form various Xaa–Cys peptide bonds, including the difficult Val–Cys junction. Facile synthesis of the Weinreb amide linkers allows easy access to this new type of peptide thioester precursor by standard Fmoc solid phase synthesis.

α-Oxo aldehyde-based bioconjugation chemistry has been widely explored in peptide and protein modifications for various applications in biomedical research during the past decades. The generation of α-oxo aldehyde via sodium periodate oxidation is usually limited to the N-terminus of a target protein. Internal-site functionalization of proteins with the α-oxo aldehyde handle has not been achieved yet. Herein we report a novel method for site-specific peptide and protein modification using synthetically or genetically incorporated thiazolidine-protected α-oxo aldehyde. Efficient unmasking of the aldehyde was achieved by silver ion-mediated hydrolysis of thiazolidine under mild conditions for the first time. A model peptide and a recombinant protein were used to demonstrate the utility of this new method, which were site-specifically modified by oxime ligation with an oxyamine-functionalized peptide labeling reagent. Therefore, our current method has enriched the α-oxo aldehyde synthetic tool box in peptide and protein bioconjugation chemistry and holds great potential to be explored in novel applications in the future.

Palladium-catalyzed acetoxylation of the primary γ-C(sp3)–H bonds in the amino acids Val, Thr, and Ile was achieved using a newly discovered 5-methylisoxazole-3-carboxamide directing group. The γ-acetoxylated α-amino acid derivatives could be easily converted to γ-mercapto amino acids, which are useful for native chemical ligation (NCL). The first application of NCL at isoleucine in the semisynthesis of a Xenopus histone H3 protein was also demonstrated.

Herein we report a novel enzymatic bioconjugation method to prepare peptide dendrimers. Under the catalysis of a newly discovered peptide ligase, butelase 1, peptide dendrimers of di-, tetra-, and octabranches were successfully synthesized using thiodepsipeptides as acyl donors for ligation with lysyl dendrimeric scaffolds. The efficient assembly of the highly clustered dendrimeric structure highlighted the versatility of butelase 1. We also showed that our synthetic antibacterial peptide dendrimers containing an RLYR motif are highly potent and broadly active against antibiotic-resistant strains.

Using a genetically incorporated azidonorleucine for ubiquitin installation, we prepared multi-milligram quantities of H2AK119ub (ubH2A). With a native isopeptide linkage, the synthetic ubH2A was used to study the activity of deubiquitinases and crosstalk between H2A ubiquitination and H3K36 methylation in the context of chemically defined mononucleosomes.

Here we report a new site-specific conjugation strategy to modify proteins via thiazolidine ligation. Proteins harbouring a 1,2-aminothiol moiety introduced by amber codon suppression technology could be modified chemoselectively with aldehyde-functionalized reagents, such as a biotin-labeled peptide or ubiquitin, under mild conditions to yield homogeneous biotinylated or ubiquitinated products.

Using a recently discovered peptide ligase, butelase 1, we developed a novel method to access protein thioesters in good yield. We successfully combined it with native chemical ligation and sortase-mediated ligation in tandem for protein C-terminal labeling and dual-terminal labeling to exploit the orthogonality of these three ligation methods.

New bidentate auxiliaries derived from the isoxazole-3-carboxamide and oxazole-4-carboxamide moieties were used for Pd-catalyzed C(sp3)–H bond activation. The results show that, when placed on a primary amine compound, 5-methylisoxazole-3-carboxamide (MICA) directs Pd-catalyzed activation of inert γ-C(sp3)–H bonds for C–C bond formation. Selective and efficient arylation and alkylation of several α-aminobutanoic acid derivatives led to various γ-substituted non-natural amino acids. The MICA directing group can be conveniently removed and recovered under very mild conditions.

A robust chemical ubiquitination method was developed. The method employed a genetically incorporated azidonorleucine as an orthogonal lysine precursor for the installation of a Gly residue bearing an Nα-auxiliary which mediated the ligation between ubiquitin(1–75)-thioester and the target protein. To demonstrate our methodology, a model protein, K48-linked diubiquitin, was synthesized with an overall yield of 35%.

An efficient solid phase synthetic protocol for salicylaldehyde ester peptides is reported. With a Ser or Thr at the N-terminus, these salicylaldehyde ester peptides can be easily converted to Ser/Thr containing cyclic peptides.

Peptides carrying a C-terminal 2-pyrrolidinemethanethiol (PMT) unit were synthesized using 9-fluorenylmethoxycarbonyl (Fmoc) solid-phase peptide synthesis (SPPS), and were shown to ligate efficiently with cysteinyl-peptides. This novel PMT-mediated ligation tolerated many different C-terminal residues and was successfully applied to a one-pot N-to-C sequential ligation reaction and the semi-synthesis of lysine 16 acetylated histone H4, demonstrating the utility of the method in peptide and protein synthesis.

A novel N- to C-terminus sequential chemical ligation approach has been developed for protein synthesis. Key to this strategy is the relative stability of the N,N-bis(2-mercaptoethyl)amide (BMEA) to the conventional conditions of native chemical ligation. We have also found a new thiol additive for the BMEA-mediated ligation reaction. The usefulness of this approach was demonstrated in the syntheses of a medium-sized peptide and ubiquitin.

The first application of thioacid capture ligation in protein synthesis is described. Two histone H3 proteins were synthesized in which a 30 min ligation reaction gave the protein products in good yields.

With two β-mercaptoethyl groups on the N, a tertiary amide of structure 1 is always poised for intramolecular thioesterification however it flips about the C−N bond. It is shown that a peptide with such a C-terminal N,N-bis(2-mercaptoethyl)-amide (BMEA) can be used directly for native chemical ligation (NCL). These BMEA peptides are easily prepared with standard Fmoc-solid phase peptide synthesis protocols, thus giving a very convenient access to the thioester components for NCL.

The dual native chemical ligation at lysine strategy was revised by replacing the acid-labile Cbz protecting group with photolabile NVOC at the 4-mercaptolysine side chain. The optimized strategy was subsequently applied to the synthesis of K48-linked diubiquitin.

Modification on the γ-N of the PNA backbone yielded a PNA analogue with a peptoid-like side chain. We found that the length of the side chain was important in influencing the hybridization affinity of the modified PNA.

The preparation of protein bioconjugates has been largely dependent on the development of selective chemistries that are orthogonal to the diverse functionalities present in a protein. Here, we report a new method for C-terminus-directed modification of recombinant proteins. The method is based on the thioacid/azide amidation reaction. Essentially, hydrothiolytic cleavage of the thioester intermediate in protein splicing yields a recombinant protein with a unique thioacid group at the C-terminus, which is then chemoselectively amidated with an electron-poor organic azide carrying a biofunctional tag. The small ubiquitin protein was used as a model system to demonstrate the utility of this new bioconjugation method. C-terminal PEGylation or biotinylation of ubiquitin was readily achieved through amidation of ubiquitin thioacid with a sulfonazide-functionalized PEG or biotin derivative. Our data validate that thioacid/azide amidation is a mechanistically novel and practically useful method for site-selective protein modification.

A general and diastereoselective synthesis of (2S, 4S)-4-mercapto-l-lysine derivative was described. The key features of this synthesis include Zn-mediated diastereoselective Reformatsky reaction and selective reduction of methyl ester with sodium borohydride. Introduction of thiol functional group on lysine side chain proved to be appropriate for dual native chemical ligation. This methodology allows to develop various 4-substituted l-lysine derivatives.

A thiol group introduced on the γ-carbon of lysine mediates robust native chemical ligation at both the α- and ε-amines in two consecutive steps. Desulfurization then affords the final product, in which the lysine residue at the ligation site has an isopeptide bond on its side chain. The method is useful for the synthesis of proteins containing special post-translational modifications on lysine.

The first application of thioacid capture ligation in protein synthesis is described. Two histone H3 proteins were synthesized in which a 30 min ligation reaction gave the protein products in good yields.

Peptide Weinreb amide derivatives with an N-substituted mercaptoethyl group are designed as thioester precursors for native chemical ligation. We show that these amides undergo rapid ligation with a cysteinyl peptide under normal NCL conditions to form various Xaa–Cys peptide bonds, including the difficult Val–Cys junction. Facile synthesis of the Weinreb amide linkers allows easy access to this new type of peptide thioester precursor by standard Fmoc solid phase synthesis.

The dual native chemical ligation at lysine strategy was revised by replacing the acid-labile Cbz protecting group with photolabile NVOC at the 4-mercaptolysine side chain. The optimized strategy was subsequently applied to the synthesis of K48-linked diubiquitin.

Here we report a new site-specific conjugation strategy to modify proteins via thiazolidine ligation. Proteins harbouring a 1,2-aminothiol moiety introduced by amber codon suppression technology could be modified chemoselectively with aldehyde-functionalized reagents, such as a biotin-labeled peptide or ubiquitin, under mild conditions to yield homogeneous biotinylated or ubiquitinated products.

A robust chemical ubiquitination method was developed. The method employed a genetically incorporated azidonorleucine as an orthogonal lysine precursor for the installation of a Gly residue bearing an Nα-auxiliary which mediated the ligation between ubiquitin(1–75)-thioester and the target protein. To demonstrate our methodology, a model protein, K48-linked diubiquitin, was synthesized with an overall yield of 35%.

Using a recently discovered peptide ligase, butelase 1, we developed a novel method to access protein thioesters in good yield. We successfully combined it with native chemical ligation and sortase-mediated ligation in tandem for protein C-terminal labeling and dual-terminal labeling to exploit the orthogonality of these three ligation methods.

Using a genetically incorporated azidonorleucine for ubiquitin installation, we prepared multi-milligram quantities of H2AK119ub (ubH2A). With a native isopeptide linkage, the synthetic ubH2A was used to study the activity of deubiquitinases and crosstalk between H2A ubiquitination and H3K36 methylation in the context of chemically defined mononucleosomes.