Solid-phase peptide synthesis (SPPS) is a primary source of peptides for structure–function studies in development of potential clinical, agricultural, veterinary or cosmetic applications. Despite its efficiency and scalability, SPPS produces unsustainable volumes of toxic waste and non-degradable solid supports. With regulatory changes now in effect to reduce solvent use, there is a pressing need for sustainable, water-based synthesis methods. Due to the aqueous insolubility of the commonly employed Nα-Fmoc-amino acids—the primary building blocks in SPPS—establishing water-based peptide synthesis protocols is extremely challenging. Here we show that amine salts of, for example, N-methylmorpholine or N,N,N-triethanolamine and Nα-Fmoc protected amino acids are water soluble at high concentrations. Activation with a pre-formed water-soluble activating agent enables high quality SPPS on a hydrophilic and biodegradable poly-ε-lysine-based solid support in aqueous solution without characteristic side reactions. The presented methodology provides a simple avenue to sustainable, scalable water-based chemical synthesis of peptides. The growing market demand for peptides is drawing more attention to their industrial synthetic procedures, which rely on large amounts of toxic solvents. Here the authors suggest practical steps that bring fully water-based peptide synthesis closer to reality.

The invention of solid-phase peptide synthesis (SPPS) in the late 1950s marked the end of the era of laborious and technically difficult classical solution-phase assembly of these biomolecules. SPPS enabled the ready production of defined peptide sequences with unprecedented speed, reliability and scalability, revolutionising both fundamental biochemical research and therapeutic development. Over the following decades, iterative improvements in amino acid protecting group strategies, coupling reagents and instrumentation further cemented SPPS as a foundation of modern peptide science. This mini-review aims to provide a historical overview of the development and application of continuous-flow (CF) methods to SPPS, leading to its present status as the most effective means for the automated, sustainable, chemically efficient production of peptides. By integrating the principles of flow chemistry with solid-phase techniques, CF-SPPS delivers major gains in reagent economy, reaction kinetics and environmental impact. However, a major issue remains with the solid support itself. The continued reliance on resins originally optimised for batch processes and their corresponding unsuitability has restricted the full realisation of CF-SPPS’s potential. Thus, the development and implementation of next-generation polymer supports tailored specifically for CF processes represents a key frontier in the ongoing refinement of CF-SPPS.

Glucagon-like peptide receptor (GLP-1R) agonists (e.g., semaglutide, liraglutide, etc.) are efficient treatment options for people with type 2 diabetes and obesity. The manufacturing method to produce semaglutide, a blockbuster GLP-1 drug on the market, involves multistep synthesis. The large peptide has a hydrophobic fatty acid side chain that makes it sparingly soluble, and its handling, purification, and large-scale production difficult. The growing demand for semaglutide that the manufacturer is not capable of addressing immediately triggered a worldwide shortage. Thus, we have developed a potential alternative analogue to semaglutide by replacing the hydrophobic fatty acid with a hydrophilic human complex-type biantennary oligosaccharide. Our novel glycoGLP-1 analogue was isolated in an ∼10-fold higher yield compared with semaglutide. Importantly, our glycoGLP-1 analogue possessed a similar GLP-1R activation potency to semaglutide and was biologically active in vivo in reducing glucose levels to a similar degree as semaglutide.

Exenatide was the first marketed GLP-1 receptor agonistfor thetreatment of type 2 diabetes. Modification to the chemical structureor the formulation has the potential to increase the stability ofexenatide. We introduced human complex-type sialyloligosaccharideto exenatide at the native Asn28 position. The synthesis was achievedusing both solid phase peptide synthesis (SPPS) and Omniligase-1-mediatedchemoenzymatic ligation. The results demonstrate that glycosylationincreases the proteolytic stability of exenatide while retaining itsfull biological activity.

Antimicrobial peptides (AMPs) are host defense peptides, and unlike conventional antibiotics, they possess potent broad spectrum activities and, induce little or no antimicrobial resistance. They are attractive lead molecules for rational development to improve their therapeutic index. Our current studies examined dimerization of the de novo designed proline-rich AMP (PrAMP), Chex1-Arg20 hydrazide, via C-terminal thiol addition to a series of bifunctional benzene or phenyl tethers to determine the effect of orientation of the peptides and linker length on antimicrobial activity. Antibacterial assays confirmed that dimerization per se significantly enhances Chex1-Arg20 hydrazide action. Greatest advantage was conferred using perfluoroaromatic linkers (tetrafluorobenzene and octofluorobiphenyl) with the resulting dimeric peptides 6 and 7 exhibiting potent action against Gram-negative bacteria, especially the World Health Organization's critical priority-listed multidrug-resistant (MDR)/extensively drug-resistant (XDR) Acinetobacter baumannii as well as preformed biofilms. Mode of action studies indicated these lead PrAMPs can interact with both outer and inner bacterial membranes to affect the membrane potential and stress response. Additionally, 6 and 7 possess potent immunomodulatory activity and neutralise inflammation via nitric oxide production in macrophages. Molecular dynamics simulations of adsorption and permeation mechanisms of the PrAMP on a mixed lipid membrane bilayer showed that a rigid, planar tethered dimer orientation, together with the presence of fluorine atoms that provide increased bacterial membrane interaction, is critical for enhanced dimer activity. These findings highlight the advantages of use of such bifunctional tethers to produce first-in-class, potent PrAMP dimers against MDR/XDR bacterial infections.

The current commercially available glucagon formulations for the treatment of severe hypoglycemia must be reconstituted immediately prior to use, owing to the susceptibility of glucagon to fibrillation and aggregation in an aqueous solution. This results in the inconvenience of handling, misuse, and wastage of this drug. To address these issues, we synthesized a glycosylated glucagon analogue in which the 25th residue (Trp) was replaced with a cysteine (Cys) and a Br-disialyloligosaccharide was conjugated at the Cys thiol moiety. The resulting analogue, glycoglucagon, is a highly potent full agonist at the glucagon receptor. Importantly, glycoglucagon exhibits markedly reduced propensity for fibrillation and enhanced thermal and metabolic stability. This novel analogue is thus a valuable lead for producing stable liquid glucagon formulations that will improve patient compliance and minimize wastage.

The role of insulin and insulin-like peptides (ILPs) in vertebrate animals is well studied. Numerous ILPs are also found in invertebrates, although there is uncertainty as to the function and role of many of these peptides. We have identified transcripts with similarity to the insulin family in the tentacle transcriptomes of the sea anemone Oulactis sp. (Actiniaria: Actiniidae). The translated transcripts showed that these insulin-like peptides have highly conserved A- and B-chains among individuals of this species, as well as other Anthozoa. An Oulactis sp. ILP sequence (IlO1_i1) was synthesized using Fmoc solid-phase peptide synthesis of the individual chains, followed by regioselective disulfide bond formation of the intra-A and two interchain disulfide bonds. Bioactivity studies of IlO1_i1 were conducted on human insulin and insulin-like growth factor receptors, and on voltage-gated potassium, sodium, and calcium channels. IlO1_i1 did not bind to the insulin or insulin-like growth factor receptors, but showed weak activity against K(V)1.2, 1.3, 3.1, and 11.1 (hERG) channels, as well as Na(V)1.4 channels. Further functional studies are required to determine the role of this peptide in the sea anemone.

The growing epidemic of diabetes means that there is a need for therapies that are more efficacious, safe, and convenient. Here, we report the efficient synthesis of a novel disulfide dimer of human insulin tethered at the N-terminus of its B-chain through placement of a cysteine residue. The resulting peptide was shown to bind to both the insulin receptor isoform B and insulin-like growth factor-1 receptor with comparable affinity to native insulin. In in vivo insulin tolerance tests, the dimer was equipotent to Actrapid insulin and possessed a sustained duration of action greater than that of Actrapid and Glargine. While the secondary structure of our dimeric insulin was similar to that of insulin, it was more resistant to proteolysis. More importantly, our analogue was produced in quantitative yield from a monomeric thiol insulin scaffold. Our results suggest that this dimer has significant potential to address the clinical needs in the treatment of diabetes.

In the fruit fly Drosophila melanogaster, there are eight insulin-like peptides (DILPs) with DILPs 1-7 interacting with a sole insulin-like receptor tyrosine kinase (DInR) while DILP8 interacts with a single G protein-coupled receptor (GPCR), Lgr3. Loss-of-function dilp mutation studies show that the neuropeptide DILP2 has a key role in carbohydrate and lipid metabolism as well as longevity and reproduction. A better understanding of the processes whereby DILP2 mediates its specific actions is required. Consequently we undertook to prepare DILP2 as part of a larger, detailed structure-function relationship study. Use of our well-established insulin-like peptide synthesis protocol that entails separate solid phase assembly of each of the A- and B-chains with selective cysteine S-protection followed by sequential S-deprotection and simultaneous disulfide bond formation produced DILP2 in good overall yield and high purity. The synthetic DILP2 was shown to induce significant DInR phosphorylation and downstream signalling, with it being more potent than human insulin. This peptide will be a valuable tool to provide further insights into its binding to the insulin receptor, the subsequent cell signalling and role in insect metabolism.

Cyclotides are plant-derived, gene-encoded, circular peptides with a range of host-defense functions, including insecticidal activity. They also have potential as pharmaceutical scaffolds and understanding their biosynthesis is important to facilitate their large-scale production. Insights into the biosynthesis of cyclotides are emerging but there are still open questions, particularly regarding the influence of the structure of the precursor proteins on processing/biosynthetic pathways. The precursor protein of kalata B1, encoded by the plant Oldenlandia affinis, contains N- and C-terminal propeptides that flank the mature cyclotide domain. The C-terminal region (ctr) is important for the cyclization process, whereas the N-terminal repeat (ntr) has been implicated in vacuolar targeting. In this study we examined the structure and folding of various truncated constructs of the ntr coupled to the mature domain of kalata B1. Despite the ntr having a well-defined helical structure in isolation, once coupled to the natively folded mature domain there is no evidence of an ordered structure. Surprisingly, the ntr appears to be highly disordered and induces self-association of the precursor. This self-association might be associated with the role of the ntr as a vacuolar-targeting signal, as previously shown for unrelated storage proteins.

The adipose tissue participates in the regulation of energy homeostasis as an important endocrine organ that secretes a number of biologically active adipokines, including adiponectin. Recently we developed and characterized a first-in-class peptide-based adiponectin receptor agonist by using in vitro and in vivo models of glioblastoma and breast cancer (BC). In the current study, we further explored the effects of peptide ADP355 in additional cellular models and found that ADP355 inhibited chronic myeloid leukemia (CML) cell proliferation and renal myofibroblast differentiation with mid-nanomolar IC50 values. According to molecular modeling calculations, ADP355 was remarkably flexible in the global minimum with a turn present in the middle of the peptide. Considering these structural features of ADP355 and the fact that adiponectin normally circulates as multimeric complexes, we developed and tested the activity of a linear branched dimer (ADP399). The dimer exhibited approximately 20-fold improved cellular activity inhibiting K562 CML and MCF-7 cell growth with high pM-low nM relative IC50 values. Biodistribution studies suggested superior tissue dissemination of both peptides after subcutaneous administration relative to intraperitoneal inoculation. After screening of a 397-member adiponectin active site library, a novel octapeptide (ADP400) was designed that counteracted 10-1000 nM ADP355- and ADP399-mediated effects on CML and BC cell growth at nanomolar concentrations. ADP400 induced mitogenic effects in MCF-7 BC cells perhaps due to antagonizing endogenous adiponectin actions or acting as an inverse agonist. While the linear dimer agonist ADP399 meets pharmacological criteria of a contemporary peptide drug lead, the peptide showing antagonist activity (ADP400) at similar concentrations will be an important target validation tool to study adiponectin functions.

Relaxin, a heterodimeric polypeptide hormone, is a key regulator of collagen metabolism and multiple vascular control pathways in humans and rodents. Its actions are mediated via its cognate G-protein-coupled receptor, RXFP1 although it also "pharmacologically" activates RXFP2, the receptor for the related, insulin-like peptide 3 (INSL3), which has specific actions on reproduction and bone metabolism. Therefore, experimental tools to facilitate insights into the distinct biological actions of relaxin and INSL3 are required, particularly for studies of tissues containing both RXFP1 and RXFP2. Here, we chemically functionalized human (H2) relaxin, the RXFP1-selective relaxin analog H2:A(4-24)(F23A), and INSL3 to accommodate a fluorophore without marked reduction in binding or activation propensity. Chemical synthesis of the two chains for each peptide was followed by sequential regioselective formation of their three disulfide bonds. Click chemistry conjugation of Cy5.5 at the B-chain N-terminus, with conservation of the disulfide bonds, yielded analogs displaying appropriate selective binding affinity and ability to activate RXFP1 and/or RXFP2 in vitro. The in vivo biological activity of Cy5.5-H2 relaxin and Cy5.5-H2:A(4-24)(F23A) was confirmed in mice, as acute intracerebroventricular (icv) infusion of these peptides (but not Cy5.5-INSL3) stimulated water drinking, an established behavioral response elicited by central RXFP1 activation. The central distribution of Cy5.5-conjugated peptides was examined in mice killed 30 min after infusion, revealing higher fluorescence within brain tissue near-adjacent to the cerebral ventricle walls relative to deeper brain areas. Production of fluorophore-conjugated relaxin family peptides will facilitate future pharmacological studies to probe the function of H2 relaxin/RXFP1 and INSL3/RXFP2 signaling in vivo while tracking their distribution following central or peripheral administration.

Numerous methods have been developed for the formation of disulfide bonds in recombinant DNA-derived and chemically synthesized peptides and proteins, but only a few have found widespread acceptance. The choice of method(s) for formation of disulfide in synthetic peptides and proteins needs to be tailored for each individual polypeptide in such a way so that the reaction conditions are selective, efficient, and safe and give the maximum yield. Here we describe the sequential formation of three disulfide bonds regioselectively which has been optimized for the synthesis of two-chained, heterodimeric polypeptide members of the insulin-relaxin superfamily.

Background:The chemical synthesis of multi-disulfide bonded heterodimeric peptides such as insulin has long been of significant scientific and commercial interest as well as a major challenge.The development of improved protocols which includes regioselective disulfide bond formation has greatly advanced the capacity to prepare and study insulin-like peptides including canine relaxin, an important regulator of parturition and indicator of canine maternal health.Methods: Separate, efficient solid phase synthesis of the two constituent chains (24 residue A and 35 residue B) was followed by stepwise formation of each of the three disulfide bonds, one intra within the A-chain and two interchain, by oxidation, thiolysis and iodolysis respectively.Results: Synthetic canine relaxin having a total of 59 residues was prepared in good overall yield and shown by several criteria to be highly purified.The peptide was shown to be less potent than human relaxin (H2 relaxin) in binding to and activating the human relaxin receptor, RXFP1, in transfected cells.A circular dichrosim spectroscopic analysis showed that the canine relaxin also possessed significantly less secondary structure compared to H2 relaxin which may account for its reduced activity. Conclusions:The synthetic protocols developed in our laboratory enabled the successful preparation of the complex, small insulinlike protein, canine relaxin.This will, in turn, allow a detailed study of both the tertiary conformation of this peptide and its role in canine reproduction.

Gonococcal growth inhibitor 1 (GGI-1) is a 44-residue peptide with potent anti-Legionella activity. It has been isolated from Staphylococcus haemolyticus but, to date, its chemical synthesis has not been reported. Acquisition of this peptide via this means would enable a more detailed examination of its antimicrobial properties. However, its synthesis represents a significant challenge because of two predicted "difficult sequences" within the peptide. Its successful solid-phase assembly is reported in this paper, and was accomplished by use of simple palliative measures including the introduction of a single pseudo-proline isostere in order to counteract on-resin aggregation. The peptide had moderate antimicrobial activity against Escherichia coli but was inactive against another Gram-negative bacterium and two Gram-positive bacteria (Bacillus species). It had significant haemolytic activity, with a H50 (concentration of peptide that causes 50 % haemolysis) of 20 and 125 μM for two blood samples from different donors. An alternative therapeutic index to that proposed for GGI-1 in a recent publication is proposed.

INSL3 is a member of the insulin-IGF-relaxin superfamily and plays a key role in male fetal development and in adult germ cell maturation. It is the cognate ligand for RXFP2, a leucine-rich repeat containing G-protein coupled receptor. To date, and in contrast to our current knowledge of the key structural features that are required for the binding of INSL3 to RXFP2, comparatively little is known about the key residues that are required to elicit receptor activation and downstream cell signaling. Early evidence suggests that these are contained principally within the A-chain. To further explore this hypothesis, we have undertaken an examination of the functional role of the intra-A-chain disulfide bond. Using solid-phase peptide synthesis together with regioselective disulfide bond formation, two analogs of human INSL3 were prepared in which the intra-chain disulfide bond was replaced, one in which the corresponding Cys residues were substituted with the isosteric Ser and the other in which the Cys were removed altogether. Both of these peptides retained nearly full RXFP2 receptor binding but were devoid of cAMP activity (receptor activation), indicating that the intra-A-chain disulfide bond makes a significant contribution to the ability of INSL3 to act as an RXFP2 agonist. Replacement of the disulfide bond with a metabolically stable dicarba bond yielded two isomers of INSL3 that each exhibited bioactivity similar to native INSL3. This study highlights the critical structural role played by the intra-A-chain disulfide bond of INSL3 in mediating agonist actions through the RXFP2 receptor.

Venom has only been recently discovered to be a basal trait of the Anguimorpha lizards. Consequently, very little is known about the timings of toxin recruitment events, venom protein molecular evolution, or even the relative physical diversifications of the venom system itself. A multidisciplinary approach was used to examine the evolution across the full taxonomical range of this ∼130 million-year-old clade. Analysis of cDNA libraries revealed complex venom transcriptomes. Most notably, three new cardioactive peptide toxin types were discovered (celestoxin, cholecystokinin, and YY peptides). The latter two represent additional examples of convergent use of genes in toxic arsenals, both having previously been documented as components of frog skin defensive chemical secretions. Two other novel venom gland-overexpressed modified versions of other protein frameworks were also recovered from the libraries (epididymal secretory protein and ribonuclease). Lectin, hyaluronidase, and veficolin toxin types were sequenced for the first time from lizard venoms and shown to be homologous to the snake venom forms. In contrast, phylogenetic analyses demonstrated that the lizard natriuretic peptide toxins were recruited independently of the form in snake venoms. The de novo evolution of helokinestatin peptide toxin encoding domains within the lizard venom natriuretic gene was revealed to be exclusive to the helodermatid/anguid subclade. New isoforms were sequenced for cysteine-rich secretory protein, kallikrein, and phospholipase A(2) toxins. Venom gland morphological analysis revealed extensive evolutionary tinkering. Anguid glands are characterized by thin capsules and mixed glands, serous at the bottom of the lobule and mucous toward the apex. Twice, independently this arrangement was segregated into specialized serous protein-secreting glands with thick capsules with the mucous lobules now distinct (Heloderma and the Lanthanotus/Varanus clade). The results obtained highlight the importance of utilizing evolution-based search strategies for biodiscovery and emphasize the largely untapped drug design and development potential of lizard venoms.

Solid-phase peptide synthesis has been refined to a stage where efficient preparation of long and complex peptides is now achievable. However, the postsynthesis handling of poorly soluble peptides often remains a significant hindrance to their purification and further use. Several synthetic schemes have been developed for the preparation of such peptides containing modifications to aid their solubility. However, these require the use of complex chemistry or yield non-native sequences. We describe a simple approach based on the use of penta-lysine "tags" that are linked to the C-terminus of the peptide of interest via a base-labile linker. After ready purification of the now freely solubilized peptide, the "tag" is removed by simple, brief base treatment giving the native sequence in much higher overall yield. The applicability of the method was demonstrated by the novel preparation of insulin glargine via solid-phase synthesis of each of the two chains--including the notoriously poorly soluble A-chain--followed by their combination in solution via regioselective disulfide bond formation. At the conclusion of the chain combination, the solubilizing peptide tag was removed from the A-chain to provide synthetic human glargine in nearly 10% overall yield. This approach should facilitate the development of new insulin analogues as well as be widely applicable to the improved purification and acquisition of otherwise poorly soluble synthetic peptides.

Successful methods for the chemical assembly of insulin-like peptides allow the detailed study of their structure and function relationships. However, the two-chain, three-disulfide bond structure of this family of peptides, which includes relaxin, has long represented a significant challenge with respect to their chemical synthesis. Early efforts involved the random combination of the two synthetic S-reduced chains under oxidizing conditions to spontaneously form the three disulfide bonds. Such an approach, while generally effective for native sequences, is critically dependent upon the presence of intact secondary structures within the individual chains which guide the subsequent folding and oxidation pathway. This limitation prevents the use of this approach for the preparation of analogs in which these secondary elements are either absent or modified. Nowadays, the use of highly efficient solid-phase peptide synthesis methodologies together with selective S -thiol-protecting groups allows the acquisition of individual chains that can be combined by effective sequential chemically directed formation of each of the three disulfide bonds. These approaches have allowed the high-yield assembly of an array of insulin-like peptides which, in turn, has provided considerable and valuable structural and biological information.

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