There has been a recent renaissance in the use of peptides as therapeutic agents across a range of indications, sparking significant demand for the development of sustainable and cost-effective alternatives to solid-phase peptide synthesis (SPPS) for the production of these molecules, particularly in the pharmaceutical industry. While tag-assisted peptide synthesis (TAPS) has offered promise, this methodology cannot be routinely used to assemble longer peptide targets (>20 residues), limiting its utility for most peptide therapeutics. Fragment condensation of side-chain-protected peptides using coupling reagents is typically used to prepare larger targets, but this approach usually leads to unacceptable levels of epimerization without significant optimization. Herein, we report an efficient platform for the synthesis of pharmaceutically relevant peptides through direct aminolysis of peptide aryl selenoesters generated via TAPS. Notably, this novel ligation method circumvents the limitations of peptide length associated with TAPS, leads to minimal epimerization, and significantly reduces reagent and solvent use, making it attractive from an environmental standpoint. By integrating the aryl selenoester aminolysis ligation (ASAL) into the TAPS workflow, the convergent synthesis of several therapeutic peptides of increasing complexity was successfully accomplished, including osteoporosis drug teriparatide (34 residues), sulfated tsetse fly-derived thrombin-inhibiting anticoagulant TTI (32 residues), and tirzepatide (39 residues), used for the treatment of type 2 diabetes and weight management. When used in concert with TAPS, the ASAL reaction developed here can serve as a robust method for the ligation-based assembly of tagged peptides, creating a scalable route to access peptide-based therapeutics across academia and industry with a low environmental impact.

Peptides of the adipokinetic hormone family are responsible for metabolic roles in insects, regulating release of energy metabolites from the fat body. We report on adipokinetic hormone octapeptide sequences bearing a rarely identified post-translational modification (sulfation of a threonine residue) in two beetle subfamilies (Cetoniinae and Dynastinae) of the large superfamily of Scarabaeoidea (dung beetles, rhinoceros beetles and flower beetles), and in a bug species (family Coreidae). In the cetonids Pachnoda sinuata, Dicronorhina derbyana derbyana, Tropinota hirta, Protaetia cuprea, Cetonia aurata and Oxytherea funesta sulfated Pacsi-AKH is found (pQINLTsTGW amide), while sulfated Penid-AKH (pQVNISsTGW amide) occurs in the dynastid beetles Pentodon idiota, Xylotrupes gideon and Syrichthodontus spurius. Sulfated Schgr-AKH-II (pQLNFSsTGW amide) is found in the twig wilter Holopterna alata. Sequence elucidation was achieved by mass spectrometry, however, due to the labile nature of the sulfate group under mass spectrometric conditions, the modified amino acid could not be easily identified. Edman degradation and comparative mass spectrometry evaluations with synthetic sulfopeptide standards were therefore employed for sequence validation. This type of sulfation was previously only reported present on the protein backbone of very few proteins from vertebrates (including humans), a mollusc and a protozoan parasite.

Peptide hormone signaling coordinates plant growth and osmotic stress responses, yet how the transition between these responses is regulated remains poorly understood. Here, we investigated the function of the rice PLANT PEPTIDES CONTAINING SULFATED TYROSINE 8 (OsPSY8) peptide in osmotic stress responses. OsPSY8 was predominantly expressed in root tissues under non-stress conditions, with preferential expression in lateral roots where it promoted root growth. Osmotic stress rapidly reduced OsPSY8 expression in roots through the OsWRKY24 transcription factor. Loss-of-function ospsy8 mutants exhibited enhanced osmotic stress tolerance, whereas OsPSY8 overexpression increased osmotic stress susceptibility. Transcriptomic analyses revealed that disruption of OsPSY8 activated stress-responsive pathways, including those associated with lignin biosynthesis, compatible solute production, cell wall remodeling, and reactive oxygen species (ROS) scavenging, and was accompanied by increased lignin accumulation in roots. In contrast, overexpression of OsPSY8 resulted in maintenance of growth-associated transcriptional programs while suppressing stress-responsive pathways under osmotic stress. Together, these findings identify OsPSY8 as an important regulator of the transition from growth to stress adaptation in rice and suggest that stress-induced repression of PSY signaling is required to disengage growth programs and activate adaptive responses during osmotic stress. Significance Statement:Crop survival during drought depends on the ability to transition from growth to stress adaptation. Plant peptide hormones have emerged as important regulators of this critical transition, highlighting the importance of investigating their roles and potential for improving crop resilience. We show that a rice peptide hormone regulates this transition. Under non-stress conditions, this peptide hormone, predominantly expressed in rice roots, promotes root growth while suppressing stress responses. During osmotic stress, expression of the peptide hormone decreases, resulting in activation of stress-responsive pathways, such as lignin biosynthesis and reactive oxygen species scavenging. These findings demonstrate that a peptide hormone coordinates the balance between growth and stress adaptation in rice, with broader implications for understanding and improving crop resilience.

An iterative one-pot peptide native chemical ligation (NCL)-desulfurisation strategy, featuring 7-diethylamino-3-methyl coumarin (DEAMC) as an orthogonal protecting group for cysteine, is described. We show that selective desulfurisation of unprotected cysteine residues can be achieved in the presence of DEAMC-protected cysteine residues, allowing subsequent DEAMC photodeprotection and native chemical ligation to be performed without purification. The efficiency of this approach was exemplified through the one-pot synthesis of a 60-residue mucin-1 peptide.

Tuberculosis (TB), caused by infection with the bacterium Mycobacterium tuberculosis ( Mtb ), remains one of the most prevalent infectious diseases worldwide. Despite decades of dedicated efforts to develop effective prevention strategies, including vaccines, TB continues to cause significant morbidity and mortality globally. Beyond the Bacille Calmette–Guérin (BCG) vaccine, limited progress has been made to develop more effective TB vaccines. A better understanding of the immunomodulatory roles of key Mtb protein virulence factors is therefore needed for the development of more efficacious protein‐based vaccine candidates. Herein, we report a highly efficient method for the semi‐synthesis of two native mycobacterial lipoproteins and glycolipoproteins, LprA and Mpt83, respectively. Capitalising on the enhanced reactivity of peptide selenoesters as acyl donors, the 21–23 kDa homogeneously lipidated proteins could be generated in a single ligation step. The homogeneous mycobacterial lipoprotein and glycolipoprotein molecules were shown to be agonists of Toll‐like receptor 2 (TLR2) and led to the potent induction of pro‐inflammatory cytokines and chemokines in cells. Taken together, this work presents a robust semi‐synthetic platform for accessing lipoproteins involved in host–pathogen interactions that can help guide future TB vaccine design.

The development of a flow chemistry platform for the generation of modified protein targets via expressed protein ligation (EPL) is described. The flow EPL platform enables efficient ligation reactions with high recoveries of target protein products and superior reaction rates compared to corresponding batch processes. The utility of the flow EPL technology was first demonstrated through the semisynthesis of the tick-derived chemokine-binding protein ACA-01 containing two tyrosine sulfate modifications. Full-length, sulfated ACA-01 could be efficiently assembled by ligating a recombinantly expressed C-terminal protein fragment and a synthetic sulfopeptide thioester in flow. Following folding, the semisynthetic sulfoprotein was shown to exhibit potent binding to a variety of pro-inflammatory chemokines. In a second modified protein target, we employed an in-line flow EPL-photodesulfurization strategy to generate both unmodified and phosphorylated forms of human β-synuclein by fusing a recombinant protein thioester, generated through cleavage of an intein fusion protein, and a synthetic (phospho)peptide. The semisynthetic proteins were assembled in 90 min in flow, a significant improvement over corresponding batch protein assembly, and enabled access to tens of milligrams of high purity material. Flow EPL has the potential to serve as a robust technology to streamline access to homogeneously modified proteins for a variety of applications in both academia, as well as in the pharmaceutical and biotechnology sector.

Chemokines are small proteins involved in recruiting leukocytes to sites of inflammation via interactions with specific cell surface receptors. CCL22 is a chemokine known to play a critical role in inflammatory diseases such as atopic dermatitis and asthma; inhibition of this chemokine therefore represents an attractive therapeutic strategy. Herein, we describe the discovery of cyclic d-sulfopeptide inhibitors of CCL22 identified through mirror-image mRNA display with genetic reprogramming. Chemical synthesis of mirror-image d-CCL22 enabled screening of a cyclic peptide library comprised of all l-amino acids, with reprogramming of l-sulfotyrosine to mimic the presence of this post-translational modification on native chemokine receptors. Enriched macrocyclic peptides were prepared in their mirror-image d-form and assessed for binding against native l-CCL22. The most potent ligand, a plasma-stable d-cyclic peptide bearing four d-sulfotyrosine residues, exhibited nanomolar affinity for CCL22, high selectivity over other chemokines, and nanomolar inhibition of CCL22 signaling through CCR4. This work highlights the vast potential of mirror-image mRNA display technology for discovering proteolytically stable d-peptide inhibitors of protein-protein interactions relevant across a range of therapeutic indications.

Capitalizing on our previous kinetic target-guided synthesis (KTGS) involving the sulfo-click reaction to form N-acylsulfonamide-linked inhibitors in the presence of the protein-protein interaction target Mcl-1, we herein report a seleno-click approach for amide-linked inhibitors of Mcl-1. The seleno-click reaction leverages the enhanced reactivity of selenocarboxylates, enabling the templated amidation with electron-rich azides, thereby expanding the scope of KTGS. The potential of this approach is demonstrated by generating N-benzyl-5-(4-isopropylthiophenol)-2-hydroxyl nicotinamide, a known Mcl-1 inhibitor featuring an amide, via KTGS at 37 °C against Mcl-1. Notably, the seleno-click strategy was also effective at 4 °C, offering a variant for thermally sensitive biological targets.

Mild strategies for the selective modification of peptides and proteins are in demand for applications in therapeutic peptide and protein discovery, and in the study of fundamental biomolecular processes. Herein, we describe the development of an electrochemical selenoetherification (e-SE) platform for the efficient site-selective functionalization of polypeptides. This methodology utilizes the unique reactivity of the 21st amino acid, selenocysteine, to effect formation of valuable bioconjugates through stable selenoether linkages under mild electrochemical conditions. The power of e-SE is highlighted through late-stage C-terminal modification of the FDA-approved cancer drug leuprolide and assembly of a library of anti-HER2 affibody conjugates bearing complex cargoes. Following assembly by e-SE, the utility of functionalized affibodies for in vitro imaging and targeting of HER2 positive breast and lung cancer cell lines is also demonstrated.

Kinetic target-guided synthesis (KTGS) is a powerful screening approach that enables identification of small molecule modulators for biomolecules. While many KTGS variants have emerged, a majority of the examples suffer from limited throughput and a poor signal/noise ratio, hampering reliable hit detection. Herein, we present our optimized multifragment KTGS screening strategy that tackles these limitations. This approach utilizes selected reaction monitoring liquid chromatography tandem mass spectrometry for hit detection, enabling the incubation of 190 fragment combinations per screening well. Consequentially, our fragment library was expanded from 81 possible combinations to 1710, representing the largest KTGS screening library assembled to date. The expanded library was screened against Mcl-1, leading to the discovery of 24 inhibitors. This work unveils the true potential of KTGS with respect to the rapid and reliable identification of hits, further highlighting its utility as a complement to the existing repertoire of screening methods used in drug discovery.

The importance of modified peptides and proteins for applications in drug discovery, and for illuminating biological processes at the molecular level, is fueling a demand for efficient methods that facilitate the precise modification of these biomolecules. Herein, we describe the development of a photocatalytic method for the rapid and efficient dimerization and site-specific functionalization of peptide and protein diselenides. This methodology, dubbed the photocatalytic diselenide contraction, involves irradiation at 450 nm in the presence of an iridium photocatalyst and a phosphine and results in rapid and clean conversion of diselenides to reductively stable selenoethers. A mechanism for this photocatalytic transformation is proposed, which is supported by photoluminescence spectroscopy and density functional theory calculations. The utility of the photocatalytic diselenide contraction transformation is highlighted through the dimerization of selenopeptides, and by the generation of two families of protein conjugates via the site-selective modification of calmodulin containing the 21 st amino acid selenocysteine, and the C-terminal modification of a ubiquitin diselenide.

Peptide selenoesters have recently emerged as key building blocks for the ligation-based assembly of large polypeptides and proteins. Herein, we report an efficient solid-phase method for the high yielding and epimerisation-free synthesis of peptide selenoesters using a side-chain immobilisation strategy.

Herein, we describe the development and application of a novel expressed protein selenoester ligation (EPSL) methodology for the one-pot semi-synthesis of modified proteins. EPSL harnesses the rapid kinetics of ligation reactions between modified synthetic selenopeptides and protein aryl selenoesters (generated from expressed intein fusion precursors) followed by in situ chemoselective deselenization to afford target proteins at concentrations that preclude the use of traditional ligation methods. The utility of the EPSL technology is showcased through the efficient semi-synthesis of ubiquitinated polypeptides, lipidated analogues of the membrane-associated GTPase YPT6, and site-specifically phosphorylated variants of the oligomeric chaperone protein Hsp27 at high dilution.

Ants (Hymenoptera: Formicidae) are familiar inhabitants of most terrestrial environments. Although we are aware of the ability of many species to sting, knowledge of ant venom chemistry remains limited. Herein, we describe the discovery and characterization of an O-linked glycopeptide (Mg7a) as a major component of the venom of the ant Myrmecia gulosa. Electron transfer dissociation and higher-energy collisional dissociation tandem mass spectrometry were used to localize three α-N-acetylgalactosaminyl residues (α-GalNAc) present on the 63-residue peptide. To allow for functional studies, we synthesized the full-length glycosylated peptide via solid-phase peptide synthesis, combined with diselenide-selenoester ligation-deselenization chemistry. We show that Mg7a is paralytic and lethal to insects, and triggers pain behavior and inflammation in mammals, which it achieves through a membrane-targeting mode of action. Deglycosylation of Mg7a renders it insoluble in aqueous solution, suggesting a key solubilizing role of the O-glycans.

A method for the rapid one-pot iterative assembly of proteins via diselenide–selenoester ligation (DSL) chemistry is described that capitalises on a novel coumarin-based photolabile protecting group for selenocysteine.

The synthesis and thrombin inhibitory activity of eight homogeneously sulfated variants of the haemathrin proteins from tick saliva is described.

Peptide ligation chemistry has revolutionized protein science by providing access to homogeneously modified peptides and proteins. However, lipidated polypeptides and integral membrane proteins-an important class of biomolecules-remain enormously challenging to access synthetically owing to poor aqueous solubility of one or more of the fragments under typical ligation conditions. Herein we describe the advent of a reductive diselenide-selenoester ligation (rDSL) method that enables efficient ligation of peptide fragments down to low nanomolar concentrations, without resorting to solubility tags or hybridizing templates. The power of rDSL is highlighted in the efficient synthesis of the FDA-approved therapeutic lipopeptide tesamorelin and palmitylated variants of the transmembrane lipoprotein phospholemman (FXYD1). Lipidation of FXYD1 was shown to critically modulate inhibitory activity against the Na+/K+ pump.

Peptides and proteins represent an important class of biomolecules responsible for a plethora of structural and functional roles in vivo. Following their translation on the ribosome, the majority of eukaryotic proteins are post-translationally modified, leading to a proteome that is much larger than the number of genes present in a given organism. In order to understand the functional role of a given protein modification, it is necessary to access peptides and proteins bearing homogeneous and site-specific modifications. Accordingly, there has been significant research effort centered on the development of peptide ligation methodologies for the chemical synthesis of modified proteins. In this chapter we outline the discovery and development of a contemporary methodology called the diselenide-selenoester ligation (DSL) that enables the rapid and efficient fusion of peptide fragments to generate synthetic proteins. The practical aspects of using DSL for the preparation of chemically modified peptides and proteins in the laboratory is described. In addition, recent advances in the application of the methodology are outlined, exemplified by the synthesis and biological evaluation of a number of complex protein targets.

The growing interest in proteins, both in fundamental research and in drug discovery, has fuelled demand for efficient synthetic methods to access these biomolecules. Although solid-phase synthesis serves as the workhorse for accessing peptides up to 50 amino acids in length, ligation technologies have underpinned protein synthesis. Native chemical ligation (NCL) represents the most widely used method and relies on the reaction of a peptide bearing an N-terminal cysteine residue with a peptide thioester. While the seminal methodology was limited to reaction at N-terminal cysteine residues, the NCL concept has recently been extended with a view to improving reaction efficiency and scope. Specifically, the discovery that cysteine residues can be desulfurized to alanine has led to the development of a range of thiol-derived variants of the proteinogenic amino acids that can be employed in protein synthesis under a ligation–desulfurization manifold. Furthermore, a number of important technologies have been developed to access larger targets via multi-fragment assembly, including methods for latent thioester activation and orthogonal protecting group strategies. Very recently, the amino acid selenocysteine, together with selenylated proteinogenic amino acid variants, has been shown to facilitate rapid ligation with peptide selenoesters. The large rate accelerations of these ligations have enabled access to proteins on unprecedented timescales, while chemoselective deselenization chemistry renders hitherto unobtainable targets accessible. This Review highlights innovative developments that have greatly expanded the NCL concept, allowing it to serve as a rapid and efficient means of conquering more challenging synthetic protein targets in the near future.