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The international peptide community rejoiced when one of its most distinguished members, Morten Meldal of Denmark, shared the 2022 Nobel Prize in Chemistry. In fact, the regiospecific solid-phase "copper(I)-catalyzed 1,3-dipolar cycloaddition of terminal alkynes to azides" (CuACC) reaction-that formed the specific basis for Meldal's recognition-was reported first at the 17 th American Peptide Symposium held in San Diego in June 2001. The present perspective outlines intertwining conceptual and experimental threads pursued concurrently in Copenhagen and Minneapolis, sometimes by the same individuals, that provided context for Meldal's breakthrough discovery. Major topics covered include orthogonality in chemistry; the dithiasuccinoyl (Dts) protecting group for amino groups in α-amino acids, carbohydrates, and monomers for peptide nucleic acids (PNA); and poly(ethylene glycol) (PEG)-based solid supports such as PEG-PS, PEGA, and CLEAR [and variations inspired by them] for solid-phase peptide synthesis (SPPS), solid-phase organic synthesis (SPOS), and combinatorial chemistry that can support biological assays in aqueous media.

Solid-phase synthesis of cyclic, branched or side-chain-modified peptides typically involves introduction of a residue carrying a temporary side-chain protecting group that undergoes selective on-resin removal. In particular, N α -Fmoc-N ε -(4-methyltriphenylmethyl) (Mtt)-protected lysine and its shorter analogues are commercially available and extensively used in this context. Nevertheless, rapid reliable methods for on-resin removal of Mtt groups in the presence of tert-butyloxycarbonyl (Boc) groups are needed. Current commonly used conditions involve low concentrations (1-3%) of trifluoroacetic acid (TFA) in dichloromethane, albeit adjustment to each specific application is required to avoid premature removal of Boc groups or cleavage from the linker. Hence, a head-to-head comparison of several deprotection conditions was performed. The selected acids represent a wide range of acidity from TFA to trifluoroethanol. Also, on-resin removal of the N-(4-methoxytriphenylmethyl) (Mmt) and O-trityl groups (on serine) was investigated under similar conditions. The mildest conditions identified for Mtt deprotection involve successive treatments with 30% hexafluoroisopropanol (HFIP) or 30% perfluoro-tert-butanol [(CF 3 ) 3 COH] in dichloromethane (3 × 5 or 3 × 15 min, respectively), while 30% HFIP, 30% (CF 3 ) 3 COH, or 10% AcOH-20% trifluoroethanol (TFE) in CH 2 Cl 2 (3 × 5 min) as well as 5% trichloroacetic acid in CH 2 Cl 2 (3 × 2 min) enabled Mmt removal. Treatment with 1% TFA with/without 2% triisopropylsilane added (3 × 5 min), but also prolonged treatment with 30% (CF 3 ) 3 COH (5 × 15 min), led to selective deprotection of an O-Trt group on a serine residue. In all cases, the sequences also contained N-Boc or O-tBu protecting groups, which were not affected by 30% HFIP or 30% (CF 3 ) 3 COH even after a prolonged reaction time of 4 h. Finally, the optimized conditions involving HFIP or (CF 3 ) 3 COH proved applicable also for selective deprotection of a longer resin-bound peptide [i.e., Ac-Gly-Leu-Leu-Lys(Mtt)-Arg(Pbf)-Ile-Lys(Boc)-Ser(tBu)-Leu-Leu-RAM-PS] as well as allowed for an almost complete deprotection of a Dab(Mtt) residue., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

Previous optimisation studies of peptide/peptoid hybrids typically comprise comparison of structurally related analogues displaying different oligomer length and diverse side chains. The present work concerns a systematically constructed series of 16 closely related 12-mer oligomers with an alternating cationic/hydrophobic design, representing a wide range of hydrophobicity and differences in relative side-chain lengths. The aim was to explore and rationalise the structure-activity relationships within a subclass of oligomers displaying variation of three structural features: (i) cationic side-chain length, (ii) hydrophobic side-chain length, and (iii) type of residue that is of a flexible peptoid nature. Increased side-chain length of cationic residues led to reduced hydrophobicity till the side chains became more extended than the aromatic/hydrophobic side chains, at which point hydrophobicity increased slightly. Evaluation of antibacterial activity revealed that analogues with lowest hydrophobicity exhibited reduced activity against E. coli , while oligomers with the shortest cationic side chains were most potent against P. aeruginosa . Thus, membrane-disruptive interaction with P. aeruginosa appears to be promoted by a hydrophobic surface of the oligomers (comprised of the aromatic groups shielding the cationic side chains). Peptidomimetics with short cationic side chains exhibit increased hemolytic properties as well as give rise to decreased HepG2 (hepatoblastoma G2 cell line) cell viability. An optimal hydrophobicity window could be defined by a threshold of minimal hydrophobicity conferring activity toward E. coli and a threshold for maximal hydrophobicity, beyond which cell selectivity was lost.

Multidrug-resistant bacteria pose a serious threat to public health worldwide. Previously, α-peptide/β-peptoid hybrid oligomers were found to display activity against Gram-negative multidrug-resistant bacteria. In the present work, the influence of hydrophobicity, fluorination, and distribution of cationic/hydrophobic residues on antimicrobial, hemolytic, and cytotoxic properties of α-peptide/β-peptoid hybrids were investigated. An array of 22 peptidomimetics was tested. Analogues with enhanced hydrophobicity were found to exhibit increased activity against Gram-positive bacteria. Incorporation of fluorinated residues into the peptidomimetics conferred increased potency against Gram-positive bacteria, while hemolytic properties and activity against Gram-negative bacteria depended on the degree and type of fluorination. Generally, shorter oligomers were less potent than the corresponding longer analogues. However, some short analogues exhibited equal or higher antimicrobial activity. The alternating hydrophobic/cationic design proved superior to other distribution patterns of cationic side chains and hydrophobic moieties., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Solid-phase peptide synthesis (SPPS) is the method of choice for chemical synthesis of peptides. In this nonspecialist review, we describe commonly used resins, linkers, protecting groups, and coupling reagents in 9-fluorenylmethyloxycarbonyl (Fmoc) SPPS. Finally, a detailed protocol for manual Fmoc SPPS is presented.

A convenient strategy for the on-resin synthesis of macrocyclic peptides (3- to 13-mers) via intramolecular halide substitution by a diamino acid is described. The method is compatible with standard Fmoc/tBu SPPS and affords a tail-to-side-chain macrocyclic peptide featuring an endocyclic secondary amine. This functional group is still reactive toward acylation, allowing for the continuation of the synthesis. An application to the synthesis of lipidated cyclic and bicyclic antimicrobial peptides is presented.

To produce antibodies against synthetic peptides it is necessary to couple them to a protein carrier. This chapter provides a nonspecialist overview of peptide-carrier conjugation. Furthermore, a protocol for coupling cysteine-containing peptides to bovine serum albumin is outlined.

Synthetic peptides are important as drugs and in research. Currently, the method of choice for producing these compounds is solid-phase peptide synthesis. In this nonspecialist review, we describe the scope and limitations of Fmoc solid-phase peptide synthesis. Furthermore, we provide a detailed protocol for Fmoc peptide synthesis.

Mass spectrometry (MS) is well suited for analysis of the identity and purity of synthetic peptides. The sequence of a synthetic peptide is most often known, so the analysis is mainly used to confirm the identity and purity of the peptide. Here, simple procedures are described for MALDI-TOF-MS and LC-MS of synthetic peptides.

Calreticulin is a chaperone of the endoplasmic reticulum (ER) assisting proteins in achieving the correctly folded structure. Details of the binding specificity of calreticulin are still a matter of debate. Calreticulin has been described as an oligosaccharide-binding chaperone but data are also accumulating in support of calreticulin as a polypeptide binding chaperone. In contrast to mammalian immunoglobulin G (IgG), which has complex type N-glycans, chicken immunoglobulin Y (IgY) possesses a monoglucosylated high mannose N-linked glycan, which is a ligand for calreticulin. Here, we have used solid and solution-phase assays to analyze the in vitro binding of calreticulin, purified from human placenta, to human IgG and chicken IgY in order to compare the interactions. In addition, peptides from the respective immunoglobulins were included to further probe the binding specificity of calreticulin. The experiments demonstrate the ability of calreticulin to bind to denatured forms of both IgG and IgY regardless of the glycosylation state of the proteins. Furthermore, calreticulin exhibits binding to peptides (glycosylated and non-glycosylated) derived from trypsin digestion of both immunoglobulins. Additionally, calreticulin peptide binding was examined with synthetic peptides covering the IgG Cγ2 domain demonstrating interaction with approximately half the peptides. Our results show that the dominant binding activity of calreticulin in vitro is toward the polypeptide moieties of IgG and IgY even in the presence of the monoglucosylated high mannose N-linked oligosaccharide on IgY., (2011 Elsevier B.V. All rights reserved.)

Halocidin is a heterodimeric antimicrobial peptide isolated from a tunicate, Halocynthia aurantium. We used the most active of the two monomers, an 18 residue amidated peptide, as lead structure and determined the role of each amino acid with alanine scanning. The results obtained led to the synthesis of a first generation of analogues with antimicrobial activity. The selectivity towards bacterial versus mammalian cells has been explored, as well as the specificity for gram positive (Staphylococcus aureus ATCC 25923) versus gram negative bacteria (Escherichia coli ATCC 25922). GRAVY (grand average of hydropathicity) was used to analyze the results., (Copyright 2004 The Royal Society of Chemistry)

[Display omitted] Self-assembled hyaluronic acid-based nanogels are versatile drug carriers due to their biodegradable nature and gentle preparation conditions, making them particularly interesting for delivery of peptide therapeutics. This study aims to elucidate the relation between peptide structure and encapsulation in a nanogel. Key peptide properties that affect encapsulation in octenyl succinic anhydride-modified hyaluronic acid nanogels were identified as we explored the effect on nanogel characteristics using 12 peptides with varying charge and hydrophobicity. The size and surface properties of the microfluidics-assembled peptide-loaded nanogels were evaluated using dynamic light scattering, laser Doppler electrophoresis, and small angle neutron scattering. Additionally, the change in peptide secondary structure upon encapsulation in nanogels, their release from the nanogels, and the in vitro antimicrobial activity were assessed. In conclusion, the more hydrophobic peptides showed stronger binding to the nanogel carrier and localized internally rather than on the surface of the nanogel, resulting in more spherical nanogels with smoother surfaces and slower release profiles. In contrast, cationic and hydrophilic peptides localized at the nanogel surface resulting in fluffier nanogel structures and quick and more complete release in biorelevant medium. These findings emphasize that the advantages of nanogel delivery systems for different applications depend on the therapeutic peptide properties. [ABSTRACT FROM AUTHOR]

Rheumatoid arthritis (RA) is an autoimmune disease of unknown etiology. The majority of individuals with RA are positive for the disease-specific anti-citrullinated protein antibodies (ACPAs). These antibodies are primarily of cross-reactive nature, hence, the true autoantigen to ACPA remains unidentified. In this study, we analyzed the reactivity of RA sera to several post-translationally modified epitopes, in order to further characterize the specific nature of ACPAs by immunoassays. Substituting citrulline with other amino acids, e.g., D-citrulline, homo-citrulline and methyl-arginine illustrated that ACPAs are utmost specific for citrullinated targets. Collectively, these findings support that ACPAs and citrullinated targets are specific for RA, making citrulline-containing peptide targets the most effective assays for detection of ACPAs. [ABSTRACT FROM AUTHOR]

Methicillin-resistant Staphylococcus pseudintermedius (MRSP) constitutes an emerging health problem for companion animals in veterinary medicine. Therefore, discovery of novel antimicrobial agents for treatment of Staphylococcus-associated canine infections is urgently needed to reduce use of human antibiotics in veterinary medicine. In the present work, we characterized the antimicrobial activity of the peptoid D2 against S. pseudintermedius and Pseudomonas aeruginosa, which is another common integumentary pathogen in dogs. Furthermore, we performed a structure–activity relationship study of D2, which included 19 peptide/peptoid analogs. Our best compound D2D, an all d-peptide analogue, showed potent minimum inhibitory concentrations (MICs) against canine S. pseudintermedius (2–4 µg/mL) and P. aeruginosa (4 µg/mL) isolates as well as other selected dog pathogens (2–16 µg/mL). Time–kill assays demonstrated that D2D was able to inhibit MRSP in 30 min at 1× MIC, significantly faster than D2. Our results suggest that at high concentrations D2D is rapidly lysing the bacterial membrane while D2 is inhibiting macromolecular synthesis. We probed the mechanism of action at sub-MIC concentrations of D2, D2D, the l-peptide analog and its retro analog by a macromolecular biosynthesis assay and fluorescence spectroscopy. Our data suggest that at sub-MIC concentrations D2D is membrane inactive and primarily works by cell wall inhibition, while the other compounds mainly act on the bacterial membrane. [ABSTRACT FROM AUTHOR]

Although cyclic peptide structures are usually investigated as highly constrained scaffolds, cyclic antimicrobial peptides of natural origin often feature flexible residues. Hereby we report our findings concerning a structure-activity study conducted on a model sequence by replacing a glycine residue with a variety of flexible residues (i.e. ω-amino and α,ω-diamino acids). The resulting library has been tested for antimicrobial activity against a wide range of clinically relevant pathogens as well as for toxicity to red blood cells. Circular dichroism and molecular modelling have been used to study changes in conformation. Increments as high as 16-fold in antimicrobial activity (as effective as lipidation) and >2-fold in haemolytic EC 50 values were observed. Interestingly, secondary structures can be stabilized by increasing, rather than decreasing, ring flexibility. [ABSTRACT FROM AUTHOR]

Abstract: Calreticulin is a molecular chaperone with specificity for polypeptides and N-linked monoglucosylated glycans. In order to determine the specificity of polypeptide binding, the interaction of calreticulin with polypeptides was investigated using synthetic peptides of different length and composition. A large set of available synthetic peptides (n =127) was tested for binding to calreticulin and the results analysed by multivariate data analysis. The parameter that correlated best with binding was hydrophobicity while β-turn potential disfavoured binding. Only hydrophobic peptides longer than 5 amino acids showed binding and a clear correlation with hydrophobicity was demonstrated for oligomers of different hydrophobic amino acids. Insertion of hydrophilic amino acids in a hydrophobic sequence diminished or abolished binding. In conclusion our results show that calreticulin has a peptide-binding specificity for hydrophobic sequences and delineate the fine specificity of calreticulin for hydrophobic amino acid residues. [Copyright &y& Elsevier]