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In recent years, peptides have gained more success as therapeutic compounds. Nowadays, the preferred method to obtain peptides is solid-phase peptide synthesis (SPPS), which does not respect the principles of green chemistry due to the large number of toxic reagents and solvents used. The aim of this work was to research and study an environmentally sustainable solvent able to replace dimethylformamide (DMF) in fluorenyl methoxycarbonyl (Fmoc) solid-phase peptide synthesis. Herein, we report the use of dipropyleneglycol dimethylether (DMM), a well-known green solvent with low human toxicity following oral, inhalant, and dermal exposure and that is easily biodegradable. Some tests were needed to evaluate its applicability to all the steps of SPPS, such as amino acid solubility, resin swelling, deprotection kinetics, and coupling tests. Once the best green protocol was established, it was applied to the synthesis of different length peptides to study some of the fundamental parameters of green chemistry, such as PMI (process mass intensity) and the recycling of solvent. It was revealed that DMM is a valuable alternative to DMF in all steps of solid-phase peptide synthesis. [ABSTRACT FROM AUTHOR]

In screening the venoms of various snake species, we found that the venom of the Madagascar cat-eyed snake Madagascarophis colubrinus competes with α-bungarotoxin for binding to the nicotinic acetylcholine receptor from Torpedo californica. Using liquid chromatography, a peptide that inhibits the binding of α‑bungartoxin to this receptor was isolated from the venom and named macoluxin. The amino acid sequence of this 23-amino acid peptide was determined by automatic Edman degradation. Comparison with amino acid sequences of known proteins showed that the macoluxin sequence is homologous to the α-helical region of the sequence of snake venom metalloproteinases. The peptide was synthesized by solid-phase peptide synthesis, and the study of its biological activity showed that it inhibits the binding of α-bungarotoxin to the Torpedo receptor with an IC50 of 47 μM. Macoluxin also reversibly inhibited acetylcholine-induced currents in the muscle-type nicotinic acetylcholine receptor. This is the first data on the presence of a peptide that can inhibit the nicotinic acetylcholine receptor in the venom of rear-fanged snakes. [ABSTRACT FROM AUTHOR]

Peptides are low-molecular-weight substances involved in numerous physiological processes, such as growth and development of the organism, stress, regulation of the emotional state, sexual behavior, and immune responses. Most peptides act as ligands for proteins, the interaction with which leads to highly selective effects. Such properties, as well as a low toxicity, allow us to consider peptides as potential effective drugs. The preparation of peptide drugs became possible at the beginning of the 20th century after the development of a method of their selective synthesis. Despite the success of the synthesis of the first peptide drugs, many issues associated with an increase in the stability, bioavailability, half-life, and the ability to move through the cell membranes remain unresolved. The review considers the historical path of development of the synthesis and production of peptides, and discusses modern approaches to the creation of peptide drugs and their application in biopharmaceutics, including in the development of original peptide drugs in Russia. [ABSTRACT FROM AUTHOR]

α-Conotoxins are well-known probes for the characterization of the various subtypes of nicotinic acetylcholine receptors (nAChRs). Identifying new α-conotoxins with different pharmacological profiles can provide further insights into the physiological or pathological roles of the numerous nAChR isoforms found at the neuromuscular junction, the central and peripheral nervous systems, and other cells such as immune cells. This study focuses on the synthesis and characterization of two novel α-conotoxins obtained from two species endemic to the Marquesas Islands, namely Conus gauguini and Conus adamsonii. Both species prey on fish, and their venom is considered a rich source of bioactive peptides that can target a wide range of pharmacological receptors in vertebrates. Here, we demonstrate the versatile use of a one-pot disulfide bond synthesis to achieve the α-conotoxin fold [Cys 1-3; 2-4] for GaIA and AdIA, using the 2-nitrobenzyl (NBzl) protecting group of cysteines for effective regioselective oxidation. The potency and selectivity of GaIA and AdIA against rat nicotinic acetylcholine receptors were investigated electrophysiologically and revealed potent inhibitory activities. GaIA was most active at the muscle nAChR (IC50 = 38 nM), whereas AdIA was most potent at the neuronal α6/3 β2β3 subtype (IC50 = 177 nM). Overall, this study contributes to a better understanding of the structure–activity relationships of α-conotoxins, which may help in the design of more selective tools. [ABSTRACT FROM AUTHOR]

Conotoxins are a class of disulfide-rich peptides found in the venom of cone snails, which have attracted considerable attention in recent years due to their potent activity on ion channels and potential for therapeutics. Among them, α-conotoxin RgIA, a 13-residue peptide, has shown great promise as a potent inhibitor of α9α10 nAChRs for pain management. In this study, we investigated the effect of substituting the naturally occurring L-type arginine at position 11 of the RgIA sequence with its D-type amino acid. Our results indicate that this substitution abrogated the ability of RgIA to block α9α10 nAChRs, but instead endowed the peptide with the ability to block α7 nAChR activity. Structural analyses revealed that this substitution induced significant alteration of the secondary structure of RgIA[11r], which consequently affected its activity. Our findings underscore the potential of D-type amino acid substitution as a promising strategy for designing novel conotoxin-based ligands targeting different types of nAChRs. [ABSTRACT FROM AUTHOR]

Modern technology has perfected the synthesis of catalysts such as zeolites and mesoporous silicas using organic structure directing agents (SDA) and their industrial use to catalyze a large variety of organic reactions within their pores. We suggest that early in prebiotic evolution, synergistic interplay arose between organic species in aqueous solution and silica formed from rocks by dynamic dissolution-recrystallization. The natural organics, for example, amino acids, small peptides, and fatty acids, acted as SDA for assembly of functional porous silica structures that induced further polymerization of amino acids and peptides, as well as other organic reactions. Positive feedback between synthesis and catalysis in the silica-organic system may have accelerated the early stages of abiotic evolution by increasing the formation of polymerized species.

In this study, using angiotensin converting enzyme (ACE) inhibitory peptides PHP1 and PHP2 as the parent peptides, the hydrophobicity and electrical properties of target polypeptides were altered by substituting amino acid residues. In addition, the potential bioactivity of these polypeptides was assessed using bioinformatics tools. Following this analysis, 19 polypeptide analogues were designed and synthesized using solid-phase synthesis, and their bioactivities were detected in vitro. The results revealed that the polypeptide analogues showed relatively high ACE inhibitory activities, PHP1A-6 (IC50=3.87 μmol/L), PHP2A-3 (IC50=3.33 μmol/L), PHP2A-4 (IC50=2.86 μmol/L), and PHP2A-7 (IC50=4.58 μmol/L) exhibited the highest ACE inhibitory activity levels, significantly higher than the parent peptides (P

Siderophores are organic ligands involved in dissolved iron (dFe) speciation in the oceans. Their study is crucial for a better understanding of the biogeochemical cycle of Fe in the marine environment, particularly in certain areas, such as the Southern Ocean, where Fe deficiency limits marine productivity. In this study, an analytical method is proposed for the extraction and pre-concentration of siderophores from seawater samples by solid phase extraction (SPE) and subsequent analysis by high-performance liquid chromatography—electrospray ionization—mass spectrometry (HPLC-ESI-MS/MS). Two siderophores were used as standards: Ferrioxamine E, a commercially available hydroxamate siderophore, and a staphyloferrin A-like compound with two citric acid units, synthesized in our laboratories by solid-phase peptide synthesis. A central composite design, considering different pH (2, 3.5, and 5) and sample loading volume (50, 125 and 200 mL) as variables, was used to optimize the extraction yield with SPE C18 cartridges. Tests were conducted on samples of artificial seawater spiked with siderophore standards. Ferrioxamine E showed high extraction yields in all tests carried out. On the contrary, the extraction of staphyloferrin A-like compound was significantly affected by both pH and loading volume.

Epimerisation is basically a chemical conversion that includes the transformation of an epimer into another epimer or its chiral partner. Epimerisation of amino acid is a side reaction that sometimes happens during peptide synthesis. It became the most avoided reaction because the process affects the overall conformation of the molecule, eventually even altering the bioactivity of the peptide. Epimerised products have a high similarity of physical characteristics, thus making it difficult for them to be purified. In regards to amino acids, epimerisation is very important in keeping the chirality of the assembled amino acids unchanged during the peptide synthesis and obtaining the desirable product without any problematic purification. In this review, we report several factors that induce epimerisation during peptide synthesis, including how to characterise and affect the bioactivities. To avoid undesirable epimerisation, we also describe several methods of suppressing the process.

In this paper we report an acid-modulated strategy for novel peptide microarray production on biosensor interfaces. We initially selected a controlled pore glass (CPG) as a support for solid-phase peptide synthesis (SPPS) to implement a chemistry that can be performed at the interface of multiple field effect transistor (FET) sensors, eventually to generate label-free peptide microarrays for protein screening. Our chemistry uses a temporary protection of the N-terminal amino function of each amino acid building block with a tert-butyloxycarbonyl (Boc) group that can be removed after each SPPS cycle, in combination with semi-permanent protection of the side chains of trifunctional amino acid residues. Such a protection scheme with a well-proven record of application in conventional, batchwise SPPS has been fine-tuned for optimal performance on CPG and, from there, translated to SPR chips that allow layer-by-layer monitoring of amino acid coupling. Our results validate this acid-modulated synthesis as a feasible approach for producing peptides in high yields and purity on flat glass surfaces, such as those in bio-FETs.

Targeted delivery of site-specific therapeutic agents is an effective strategy for osteoarthritis treatment. The lack of blood vessels in cartilage makes it difficult to deliver therapeutic agents like peptides to the defect area. Therefore, nucleus-targeting zwitterionic carbon nano-dots (CDs) have immense potential as a delivery vehicle for effective peptide delivery to the cytoplasm as well as nucleus. In the present study, nucleus-targeting zwitterionic CDs have been synthesized as delivery vehicle for peptides while also working as nano-agents towards optical monitoring of cartilage healing. The functional groups of zwitterion CDs were introduced by a single-step microwave assisted oxidation procedure followed by COL II peptide conjugation derived from Capra auricular cartilage through NHS/EDC coupling. The peptide-conjugated CDs (PCDs) allows cytoplasmic uptake within a short period of time (∼30 m) followed by translocation to nucleus after ∼24 h. Moreover, multicolor fluorescence of PCDs improves (blue, green, and read channel) its sensitivity as an optical code providing a compelling solution towards enhanced non-invasive tracking system with multifunctional properties. The PCDs-based delivery system developed in this study has exhibited superior ability to induce ex-vivo chondrogenic differentiation of ADMSCs as compared to bare CDs. For assessment of cartilage regeneration potential, pluronic F-127 based PCDs hydrogel was injected to rabbit auricular cartilage defects and potential healing was observed after 60 days. Therefore, the results confirm that PCDs could be an ideal alternate for multimodal therapeutic agents.

Acyldepsipeptides (ADEPs) are receiving more attention as prospective antimicrobial agents due to their unique mode of action and chemical properties. However, their therapeutic potential is limited by their poor pharmacokinetic properties. Chemical modifications have been successful in improving the biocompatibility and bioavailability of ADEPs. In the current study, ADEP1 was modified by introducing a disulphide linkage, replacement of the octa-2,4,6-trienoic acid (OTEA) with either adamantane (Ada) or palmitic acid (Pal), and lastly, comparing the use of D versus L amino acids. The antibacterial effects of the ADEP1 analogues were investigated in Gram-positive and Gram-negative strains using agar well diffusion and microdilution assays. Cytotoxicity was evaluated in human embryonic kidney (HEK)-293 and colon cancer (Caco-2) cells by the MTS assay. Using solid phase peptide synthesis (SPPS), the percentage yield of the synthetic peptides was increased to > 37% with > 96% purity. The anionic ADEP1 analogues demonstrated a broad-spectrum antibacterial activity. Although the ADEP1 analogues did not display the expected antibacterial activity in relation to the parent structure, they were not cytotoxic against the tested cell lines. This study proved that biocompatibility of natural ADEPs can be improved by modifying some of its chemical groups. Further studies are required to enhance the antibacterial activity of the ADEP1 analogues either by structural modifications or through supplementation of these anionic antimicrobial peptides (AMPs) with divalent metal ions.

Background: The ShK toxin from Stichodactyla helianthus has established the therapeutic potential of sea anemone venom peptides, but many lineage-specific toxin families in Actiniarians remain uncharacterised. One such peptide family, sea anemone 8 (SA8), is present in all five sea anemone superfamilies. We explored the genomic arrangement and evolution of the SA8 gene family in Actinia tenebrosa and Telmatactis stephensoni, characterised the expression patterns of SA8 sequences, and examined the structure and function of SA8 from the venom of T. stephensoni. Results: We identified ten SA8-family genes in two clusters and six SA8-family genes in five clusters for T. stephensoni and A. tenebrosa, respectively. Nine SA8 T. stephensoni genes were found in a single cluster, and an SA8 peptide encoded by an inverted SA8 gene from this cluster was recruited to venom. We show that SA8 genes in both species are expressed in a tissue-specific manner and the inverted SA8 gene has a unique tissue distribution. While the functional activity of the SA8 putative toxin encoded by the inverted gene was inconclusive, its tissue localisation is similar to toxins used for predator deterrence. We demonstrate that, although mature SA8 putative toxins have similar cysteine spacing to ShK, SA8 peptides are distinct from ShK peptides based on structure and disulfide connectivity. Conclusions: Our results provide the first demonstration that SA8 is a unique gene family in Actiniarians, evolving through a variety of structural changes including tandem and proximal gene duplication and an inversion event that together allowed SA8 to be recruited into the venom of T. stephensoni. [ABSTRACT FROM AUTHOR]

YM‐254890 and FR900359 are potent and selective inhibitors of the Gq/11‐signaling pathway. As such, they have been attractive targets for both synthesis and biological studies. Yet in spite of this effort, a versatile synthetic approach to the molecules that allows for the rapid construction of a variety of non‐natural and labelled analogs and an increase in the amount of those analogs available remains elusive. We report here a convergent building block approach to the molecules that can solve this challenge. [ABSTRACT FROM AUTHOR]

RNA is a molecule that can both store genetic information and perform catalytic reactions. This observed dualism places RNA into the limelight of concepts about the origin of life. The RNA world concept argues that life started from self‐replicating RNA molecules, which evolved toward increasingly complex structures. Recently, we demonstrated that RNA, with the help of conserved non‐canonical nucleosides, which are also putative relics of an early RNA world, had the ability to grow peptides covalently connected to RNA nucleobases, creating RNA‐peptide chimeras. It is conceivable that such molecules, which combined the information‐coding properties of RNA with the catalytic potential of amino acid side chains, were once the structures from which life emerged. Herein, we report prebiotic chemistry that enabled the loading of both nucleosides and RNAs with amino acids as the first step toward RNA‐based peptide synthesis in a putative RNA‐peptide world. [ABSTRACT FROM AUTHOR]

Short and ultra‐short peptides have been recently envisioned as excellent building blocks for the formulation of hydrogels with appealing properties. Due to its simplicity and capability to gel under physiological conditions, Fmoc‐FF (Nα‐fluorenylmethoxycarbonyl‐diphenylalanine), remains one of the most studied low molecular‐weight hydrogelators. Since its first identification in 2006, a plethora of its analogues were synthetized and investigated for the fabrication of novel supramolecular materials. Here we report a description of the Fmoc‐FF analogues in which the aromatic Fmoc group is replaced with other substituents. These analogues are distinguished into five different classes including derivatives: i) customized with solid phase peptide synthesis protecting groups; ii) containing non‐aromatic groups, iii) containing aromatic groups, iv) derivatized with metal complexes and v) containing stimuli‐responsive groups. The morphological, mechanical, and functional effects caused by this modification on the resulting material are also pointed out. [ABSTRACT FROM AUTHOR]

Though the bulk of the COVID-19 pandemic is behind, the search for effective and safe anti-SARS-CoV-2 drugs continues to be relevant. A highly pursued approach for antiviral drug development involves targeting the viral spike (S) protein of SARS-CoV-2 to prevent its attachment to the cellular receptor ACE2. Here, we exploited the core structure of polymyxin B, a naturally occurring antibiotic, to design and synthesize unprecedented peptidomimetics (PMs), intended to target contemporarily two defined, non-overlapping regions of the S receptor-binding domain (RBD). Monomers 1, 2, and 8, and heterodimers 7 and 10 bound to the S-RBD with micromolar affinity in cell-free surface plasmon resonance assays (KD ranging from 2.31 μM to 2.78 μM for dimers and 8.56 μM to 10.12 μM for monomers). Although the PMs were not able to fully protect cell cultures from infection with authentic live SARS-CoV-2, dimer 10 exerted a minimal but detectable inhibition of SARS-CoV-2 entry in U87.ACE2+ and A549.ACE2.TMPRSS2+ cells. These results validated a previous modeling study and provided the first proof-of-feasibility of using medium-sized heterodimeric PMs for targeting the S-RBD. Thus, heterodimers 7 and 10 may serve as a lead for the development of optimized compounds, which are structurally related to polymyxin, with improved S-RBD affinity and anti-SARS-CoV-2 potential. [ABSTRACT FROM AUTHOR]

In the early stage of bacterial translation, peptidyl-tRNAs frequently dissociate from the ribosome (pep-tRNA drop-off) and are recycled by peptidyl-tRNA hydrolase. Here, we establish a highly sensitive method for profiling of pep-tRNAs using mass spectrometry, and successfully detect a large number of nascent peptides from pep-tRNAs accumulated in Escherichia coli pthts strain. Based on molecular mass analysis, we found about 20% of the peptides bear single amino-acid substitutions of the N-terminal sequences of E. coli ORFs. Detailed analysis of individual pep-tRNAs and reporter assay revealed that most of the substitutions take place at the C-terminal drop-off site and that the miscoded pep-tRNAs rarely participate in the next round of elongation but dissociate from the ribosome. These findings suggest that pep-tRNA drop-off is an active mechanism by which the ribosome rejects miscoded pep-tRNAs in the early elongation, thereby contributing to quality control of protein synthesis after peptide bond formation. Peptidyl-tRNAs (pep-tRNAs) frequently dissociate from ribosome, called as pep-tRNA drop-off. But, its function remained unclear. The authors proposed a new quality control mechanism of protein synthesis by active rejection of miscoded pep-tRNAs in the early stage of translation. [ABSTRACT FROM AUTHOR]

Introduction: The fibroblast growth factor receptor (FGFR) family is highly expressed in a variety of tumor types and represents a new target for cancer therapy. Different FGFR subtype aberrations have been found to exhibit highly variable sensitivity and efficacy to FGFR inhibitors. Methods: The present study is the first to suggest an imaging method for assessing FGFR1 expression. The FGFR1-targeting peptide NOTA-PEG2-KAEWKSLGEEAWHSK was synthesized by manual solid-phase peptide synthesis and high-pressure liquid chromatography (HPLC) purification and then labeled with fluorine-18 using NOTA as a chelator. In vitro and in vivo experiments were conducted to evaluate the stability, affinity and specificity of the probe. Tumor targeting efficacy and biodistribution were evaluated by micro-PET/CT imaging in RT-112, A549, SNU-16 and Calu-3 xenografts. Results: The radiochemical purity of [18F]F-FGFR1 was 98.66% ± 0.30% (n = 3) with excellent stability. The cellular uptake rate of [18F]F-FGFR1 in the RT-112 cell line (FGFR1 overexpression) was higher than that in the other cell lines and could be blocked by the presence of excess unlabeled FGFR1 peptide. Micro-PET/CT imaging revealed a significant concentration of [18F]F-FGFR1 in RT-112 xenografts with no or very low uptake in nontargeted organs and tissues, which demonstrated that [18F]F-FGFR1 was selectively taken up by FGFR1-positive tumors. Conclusion: [18F]F-FGFR1 showed high stability, affinity, specificity and good imaging capacity for FGFR1-overexpressing tumors in vivo, which provides new application potential in the visualization of FGFR1 expression in solid tumors. [ABSTRACT FROM AUTHOR]

The emergence of proteins and their interactions with RNAs were a key step in the origin and early evolution of life. The abiotic synthesis of peptides has been limited in short amino acid length and is favored in highly alkaline evaporitic conditions in which RNAs are unstable. This environment is also inconsistent with estimated Hadean Earth. Prebiotic environments rich in boron are reportedly ideal for abiotic RNA synthesis. However, the effects of boron on amino acid polymerization are unclear. We report that boric acid enables the polymerization of amino acids at acidic and near-neutral pH levels based on simple heating experiments of amino acid solutions containing borate/boric acid at various pH levels. Our study provides evidence for the boron-assisted synthesis of polypeptides in prebiotically plausible environments, where the same conditions would allow for the formation of RNAs and interactions of primordial proteins and RNAs that could be inherited by RNA-dependent protein synthesis during the evolution of life. Prebiotic environments rich in boron have been postulated to be ideal for abiotic RNA synthesis, but the effects of boron on amino acid polymerization are unclear. Here, boric acid is shown to enable the polymerization of amino acids at acidic and near-neutral pH levels. [ABSTRACT FROM AUTHOR]

The G protein‐coupled kisspeptin receptor (GPR54 or KISS1R) is an important mediator in reproduction, metabolism and cancer biology; however, there are limited fluorescent probes or antibodies for direct imaging of these receptors in cells and intact tissues, which can help to interrogate their multiple biological roles. Herein, we describe the rational design and characterization of a new acid‐resistant BODIPY‐based amino acid (Trp‐BODIPY PLUS), and its implementation for solid‐phase synthesis of fluorescent bioactive peptides. Trp‐BODIPY PLUS retains the binding capabilities of both short linear and cyclic peptides and displays notable turn‐on fluorescence emission upon target binding for wash‐free imaging. Finally, we employed Trp‐BODIPY PLUS to prepare some of the first fluorogenic kisspeptin‐based probes and visualized the expression and localization of GPR54 receptors in human cells and in whole mouse pancreatic islets by fluorescence imaging. [ABSTRACT FROM AUTHOR]

Peptides are described as naturally occurring short chains of amino acids and offer great potential as therapeutic agents because of their target selectivity, safe, and well tolerable. Hence, there is an increased interest in peptides in pharmaceutical research and development and approximately more than 170 peptide therapeutics are currently being evaluated in clinical trials and many are being used as therapeutic drugs for various diseases including COVID‐19. The present Review article focuses on recent progress in peptide drug discovery and advancements in synthetic methodologies to enhance their stability and physiological activity of peptides and as well peptidomimetics. [ABSTRACT FROM AUTHOR]

Corroles have attracted increasing research interests in recent decades owing to their unique properties over porphyrins. However, the relatively inefficient and tedious synthetic procedures of corrole building blocks with functional groups for bioconjugation hindered their bioapplications. Herein, we report a highly efficient protocol to synthesize corrole‐peptide conjugates with good yields (up to 63 %) without using prepared corrole building blocks. By condensing two −COOH‐bearing‐dipyrromethane molecules onto an aldehyde group on resin‐bound peptide chains in a controllable manner, a series of desired products with long (up to 25 residues) and bioactive peptide chains were obtained with at most one chromatographic purification. The synthesized compounds exhibited potential applications as chelators for metal ions for biomedical applications, as building blocks for supramolecular materials, as well as targeted fluorescent probes. [ABSTRACT FROM AUTHOR]

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Endogenous peptides have various modulating effects on many physiological and pathological processes, including the processing of pain information. Hemorphins are hemoglobin-derived peptides with affinity for both opioid receptors and insulin-regulated aminopeptidase. We recently reported on the antinociceptive effects of hemophins of different lengths and amino acid composition after their supraspinal injection. The present study aims to elucidate the potential effects of locally (intraplantar) injected hemorphin VV-H7 (Val-Val-Tyr-Pro-Trp-Thr-Gln-Arg-Phe-NH2) and its analogs with one or two Arg-Gly-Asp sequence on carrageenan-induced hyperalgesia in rats. We used indomethacin as a reference anti-inflammatory analgesic. Pain thresholds and paw volume were measured before and at 1, 3, and 4 hours after intraplantar carrageenan injection using a paw pressure test and plethysmometry in male Wistar rats. The precursor peptide VV-H7 showed an antihyperalgesic effect comparable to that of indomethacin, while its peptide analogues one or two Arg-Gly-Asp sequence showed an acceleration of the antinociceptive effect, but shorter duration of antihyperalgesia and lack of efficacy against edema. [ABSTRACT FROM AUTHOR]

Currently non-proteinogenic amino acids and synthetic peptides are widely used as building blocks in the drugs design. Many of these compounds are enzyme inhibitors or antimicrobials. Secreted Clostridium histolyticum collagenase is considered as a virulence factor and thus is an attractive target for fighting microbial infection. Trypsin is a target for the treatment of digestive disorders. This study was aimed to find new inhibitors of collagenase and trypsin. The interaction of non-proteinogenic amino acids and peptides with C. histolyticum collagenase and trypsin has been evaluated by Molecular Docking followed by the measurement of enzyme inhibition by selected compounds. According to the Docking analysis, N-tert-butoxycarbonylglycyl-(S)-2-amino-3-(4-((4-fluorophenyl)-ethynyl)-phenyl)-α-alanine has to demonstrate the most effective interaction with collagenase. Thus, the synthesis of this dipeptide was carried out. Measurement of enzymes activity revealed that both the dipeptide and the amino acid included in its structure inhibit collagenase and trypsin. The amino acid inhibited collagenase with IC50 = 0.32 mM and trypsin with IC50 = 0.57 mM. The dipeptide inhibited collagenase with IC50 = 0.62 mM and trypsin with IC50 = 89 uM. Keywords: bacterial collagenase, molecular docking, inhibitor, peptide synthesis. [ABSTRACT FROM AUTHOR]

Wasp stings have become an increasingly serious public health problem because of their high incidence and mortality rates in various countries and regions. Mastoparan family peptides are the most abundant natural peptides in hornet venoms and solitary wasp venom. However, there is a lack of systematic and comprehensive studies on mastoparan family peptides from wasp venoms. In our study, for the first time, we evaluated the molecular diversity of 55 wasp mastoparan family peptides from wasp venoms and divided them into four major subfamilies. Then, we established a wasp peptide library containing all 55 known mastoparan family peptides by chemical synthesis and C-terminal amidation modification, and we systematically evaluated their degranulation activities in two mast cell lines, namely the RBL-2H3 and P815 cell lines. The results showed that among the 55 mastoparans, 35 mastoparans could significantly induce mast cell degranulation, 7 mastoparans had modest mast cell degranulation activity, and 13 mastoparans had little mast cell degranulation activity, suggesting functional variation in mastoparan family peptides from wasp venoms. Structure–function relationship studies found that the composition of amino acids in the hydrophobic face and amidation in the C-terminal region are critical for the degranulation activity of mastoparan family peptides from wasp venoms. Our research will lay a theoretical foundation for studying the mechanism underlying the degranulation activity of wasp mastoparans and provide new evidence to support the molecular design and molecular optimization of natural mastoparan peptides from wasp venoms in the future. [ABSTRACT FROM AUTHOR]

Octreotide, a somatostatin analogue, has shown its efficacy for the diagnostics and treatment of various types of cancer, i.e., in octreotide scan, as radio-marker after labelling with a radiopharmaceutical. To avoid toxicity of radio-labeling, octreotide-based assays can be implemented into magnetic resonance techniques, such as MRI and NMR. Here we used a Parahydrogen-Induced Polarization (PHIP) approach as a cheap, fast and straightforward method. Introduction of l-propargyl tyrosine as a PHIP marker at different positions of octreotide by manual Solid-Phase Peptide Synthesis (SPPS) led to up to 2000-fold proton signal enhancement (SE). Cell binding studies confirmed that all octreotide variants retained strong binding affinity to the surface of human-derived cancer cells expressing somatostatin receptor 2. The hydrogenation reactions were successfully performed in methanol and under physiologically compatible mixtures of water with methanol or ethanol. The presented results open up new application areas of biochemical and pharmacological studies with octreotide. [ABSTRACT FROM AUTHOR]

Peptide nucleic acids (PNAs) are an important class of DNA/RNA mimics that can hybridize complementary chains of nucleic acids with high affinity and specificity. Because of this property and their metabolic stability, PNAs have broad potential applications in different fields. Consisting of a neutral polyamide backbone, PNAs are prepared following the method used for peptide synthesis. In this regard, they are prepared by the sequential coupling of the protected monomers on a solid support using a similar approach to solid-phase peptide synthesis (SPPS). However, PNA synthesis is a little more challenging due to issues of the difficulty on the preparation of monomers and their solubility. Furthermore, the PNA elongation is jeopardized by intra/inter chain aggregation and side reactions. These hurdles can be overcome using different protecting group strategies on the PNA monomer, which also dictate the approach followed to prepare the oligomers. Herein, the main synthetic strategies driven by the protecting group scheme are discussed. However, there is still ample scope for further enhancement of the overall process. [ABSTRACT FROM AUTHOR]

In the chemical synthesis of conotoxins with multiple disulfide bonds, the oxidative folding process can result in diverse disulfide bond connectivities, which presents a challenge for determining the natural disulfide bond connectivities and leads to significant structural differences in the synthesized toxins. Here, we focus on KIIIA, a μ-conotoxin that has high potency in inhibiting Nav1.2 and Nav1.4. The non-natural connectivity pattern (C1—C9, C2—C15, C4—C16) of KIIIA exhibits the highest activity. In this study, we report an optimized Fmoc solid-phase synthesis of KIIIA using various strategies. Our results indicate that free random oxidation is the simplest method for peptides containing triple disulfide bonds, resulting in high yields and a simplified process. Alternatively, the semi-selective strategy utilizing Trt/Acm groups can also produce the ideal isomer, albeit with a lower yield. Furthermore, we performed distributed oxidation using three different protecting groups, optimizing their positions and cleavage order. Our results showed that prioritizing the cleavage of the Mob group over Acm may result in disulfide bond scrambling and the formation of new isomers. We also tested the activity of synthesized isomers on Nav1.4. These findings provide valuable guidance for the synthesis of multi-disulfide-bonded peptides in future studies. [ABSTRACT FROM AUTHOR]

In peptide synthesis, the issues related to poor sustainability, long reaction times and high process mass intensity (PMI) are necessary to promote actions aimed at redefining procedural aspects projected towards more sustainable synthetic processes. Herein, we report a fast, widely applicable and green solution-phase peptide synthesis (GSolPPS) via a continuous protocol using propylphosphonic anhydride T3P® as the coupling reagent and N-benzyloxycarbonyl-protecting group (Z), which is easily removed by hydrogenation. Because N,N-dimethylformamide (DMF) replacement was a priority, the iterative process was performed in EtOAc, pushing further on overall sustainability. The efficiency of the synthetic protocol in terms of conversion, racemization and reaction times allowed extending the scope of the work to the synthesis of the standard peptide Leu-enkephalin as a proof of concept. Among the various explored procedures, the one-pot protocol (Acont plus), avoiding work-ups, intermediate purification and any dispersion effect, allowed the achievement of PMI = 30 for each deprotection/coupling sequence necessary to introduce a single amino acid in the iterative process, without considering the possibility of solvent and base recovery. This value is the lowest reported for an oligopeptide synthesis protocol to date. [ABSTRACT FROM AUTHOR]

The rise of antimicrobial resistance has created an urgent need for antibiotic-alternative strategies for disease control and prevention. Host defense peptides (HDPs), which have both antimicrobial and immunomodulatory properties, are an important component of the innate immune system. A host-directed approach to stimulate the synthesis of endogenous HDPs has emerged as a promising solution to treat infections with a minimum risk for developing antimicrobial resistance. Among a diverse group of compounds that have been identified as inducers of HDP synthesis are polyphenols, which are naturally occurring secondary metabolites of plants characterized by the presence of multiple phenol units. In addition to their well-known antioxidant and anti-inflammatory activities, a variety of polyphenols have been shown to stimulate HDP synthesis across animal species. This review summarizes both the in vitro and in vivo evidence of polyphenols regulating HDP synthesis. The mechanisms by which polyphenols induce HDP gene expression are also discussed. Natural polyphenols warrant further investigation as potential antibiotic alternatives for the control and prevention of infectious diseases. [ABSTRACT FROM AUTHOR]

The efficient preparation of novel bioactive peptide drugs requires the availability of reliable and accessible chemical methodologies together with suitable analytical techniques for the full characterisation of the synthesised compounds. Herein, we describe a novel acidolytic method with application to the synthesis of cyclic and linear peptides involving benzyl-type protection. The process consists of the in situ generation of anhydrous hydrogen bromide and a trialkylsilyl bromide that acts as protic and Lewis acid reagents. This method proved to be useful to effectively remove benzyl-type protecting groups and cleave Fmoc/tBu assembled peptides directly attached to 4-methylbenzhydrylamine (MBHA) resins with no need for using mild trifluoroacetic acid labile linkers. The novel methodology was successful in synthesising three antimicrobial peptides, including the cyclic compound polymyxin B3, dusquetide, and RR4 heptapeptide. Furthermore, electrospray mass spectrometry (ESI-MS) is successfully used for the full characterisation of both the molecular and ionic composition of the synthetic peptides. [ABSTRACT FROM AUTHOR]

Cardiovascular ailments are a major cause of mortality where over 1.3 billion people suffer from hypertension leading to heart-disease related deaths. Snake venoms possess a broad repertoire of natriuretic peptides with therapeutic potential for treating hypertension, congestive heart failure, and related cardiovascular disease. We now describe several taipan (Oxyuranus microlepidotus) natriuretic peptides TNPa-e which stimulated cGMP production through the natriuretic peptide receptor A (NPR-A) with higher potencies for the rat NPR-A (rNPR-A) over human NPR-A (hNPR-A). TNPc and TNPd were the most potent, demonstrating 100- and 560-fold selectivity for rNPR-A over hNPR-A. In vivo studies found that TNPc decreased diastolic and systolic blood pressure (BP) and increased heart rate (HR) in conscious normotensive rabbits, to a level that was similar to that of human atrial natriuretic peptide (hANP). TNPc also enhanced the bradycardia due to cardiac afferent stimulation (Bezold–Jarisch reflex). This indicated that TNPc possesses the ability to lower blood pressure and facilitate cardiac vagal afferent reflexes but unlike hANP does not produce tachycardia. The 3-dimensional structure of TNPc was well defined within the pharmacophoric disulfide ring, displaying two turn-like regions (RMSD = 1.15 Å). Further, its much greater biological stability together with its selectivity and potency will enhance its usefulness as a biological tool. [ABSTRACT FROM AUTHOR]

Aeruginosins, a family of nonribosomal linear tetrapeptides discovered from cyanobacteria and sponges, exhibit in vitro inhibitory activity on various types of serine proteases. This family is characterized by the existence of the 2-carboxy-6-hydroxy-octahydroindole (Choi) moiety occupied at the central position of the tetrapeptide. Aeruginosins have attracted much attention due to their special structures and unique bioactivities. Although many studies on aeruginosins have been published, there has not yet been a comprehensive review that summarizes the diverse research ranging from biogenesis, structural characterization and biosynthesis to bioactivity. In this review, we provide an overview of the source, chemical structure as well as spectrum of bioactivities of aeruginosins. Furthermore, possible opportunities for future research and development of aeruginosins were discussed. [ABSTRACT FROM AUTHOR]

We report the site‐selective acetylation of partially protected monosaccharides using immobilized oligopeptide catalysts, which are readily accessible via solid‐phase peptide synthesis. The catalysts are able to invert the intrinsic selectivity, which was determined using N‐methylimidazole, for a variety of pyranosides. We demonstrate that the catalysts are stable for multiple reaction cycles and can be easily reused after separation from the reaction solution. The catalysts can also be used in flow without loss of reactivity and selectivity. [ABSTRACT FROM AUTHOR]

(1) Background: Malaria, a vector-borne infectious disease, is caused by parasites of the Plasmodium genus, responsible for increased extreme morbidity and mortality rates. Despite advances in approved vaccines, full protection has not yet been achieved upon vaccination, thus the development of more potent and safe immuno-stimulating agents for malaria prevention is a goal to be urgently accomplished. We have focused our research on a strategy to identify Plasmodium spp. epitopes by naturally acquired human antibodies and rodent malaria infection models immunized with site-directed non-natural antigens. (2) Methods: Some predictive algorithms and bioinformatics tools resembling different biological environments, such as phagosome-lysosome proteolytic degradation, affinity, and the high frequency of malaria-resistant and -sensitive HLA-II alleles were regarded for the proper selection of epitopes and potential testing. Each epitope's binding profile to both host cells and HLA-II molecules was considered for such initial screening. (3) Results: Once selected, we define each epitope-peptide to be synthesized in terms of size and hydrophobicity, and introduced peptide-bond surrogates and non-natural amino acids in a site-directed fashion, and then they were produced by solid-phase peptide synthesis. Molecules were then tested by their antigenic and immunogenic properties compared to human sera from Colombian malaria-endemic areas. The antigenicity and protective capacity of each epitope-peptide in a rodent infection model were examined. The ability of vaccinated mice after being challenged with P. berghei ANKA and P. yoelii 17XL to control malaria led to the determination of an immune stimulation involving Th1 and Th1/Th2 mechanisms. In silico molecular dynamics and modeling provided some interactions insights, leading to possible explanations for protection due to immunization. (4) Conclusions: We have found evidence for proposing MSP1-modified epitopes to be considered as neutralizing antibody stimulators that are useful as probes for the detection of Plasmodium parasites, as well as for sub-unit components of a site-directed designed malaria vaccine candidate. [ABSTRACT FROM AUTHOR]

Gomesin is a cationic antimicrobial peptide which is isolated from the haemocytes of the Brazilian tarantula Acanthoscurria gomesiana and can be produced chemically by Fmoc solid-phase peptide synthesis. Gomesin exhibits a range of biological activities, as demonstrated by its toxicity against therapeutically relevant pathogens such as Gram-positive or Gram-negative bacteria, fungi, cancer cells, and parasites. In recent years, a cyclic version of gomesin has been used for drug design and development as it is more stable than native gomesin in human serum and can penetrate and enter cancer cells. It can therefore interact with intracellular targets and has the potential to be developed as a drug lead for to treat cancer, infectious diseases, and other human diseases. This review provides a perspective on the discovery, structure–activity relationships, mechanism of action, biological activity, and potential clinical applications of gomesin. [ABSTRACT FROM AUTHOR]

Protein‐protein interactions (PPIs) involved in several diseases are predominantly mediated through short helical peptides. Unlike small molecules, due to the extended surface, the chemically constrained peptides with proper helical conformation can modulate the PPIs and thus contribute to the future drug developments. Several model systems have been introduced in the past to induce the helical structure in short peptides. Incorporating the unnatural constraints by various protocols has been excellent in biasing helical conformation and improving the proteolytic stability and the cell permeability of peptides. Hydrogen‐bond surrogate (HBS) is one of such well‐known protocols to mimic helical surfaces in numerous peptide sequences. Unlike 'Stapled' peptides, HBS‐model replaces the main‐chain i+4→i hydrogen bonding interaction with a covalent surrogate and retains the side‐chain functionality intact, which is beneficial for targeting multi‐faced PPIs. This review focuses on the chemistry behind the development of the HBS‐stabilized peptides, its biophysical characterizations, and numerous biological applications. Furthermore, this review will provide a broad overview of the HBS‐model to attract chemical biologists to target multifaced 'difficult' PPIs. [ABSTRACT FROM AUTHOR]

Macrocyclisation provides a means of stabilising the conformation of peptides, often resulting in improved stability, selectivity, affinity, and cell permeability. In this work, a new approach to peptide macrocyclisation is reported, using a cyanobenzothiazole‐containing amino acid that can be incorporated into peptides by both in vitro translation and solid phase peptide synthesis, meaning it should be applicable to peptide discovery by mRNA display. This cyclisation proceeds rapidly, with minimal by‐products, is selective over other amino acids including non N‐terminal cysteines, and is compatible with further peptide elaboration exploiting such an additional cysteine in bicyclisation and derivatisation reactions. Molecular dynamics simulations show that the new cyclisation group is likely to influence the peptide conformation as compared to previous thioether‐based approaches, through rigidity and intramolecular aromatic interactions, illustrating their complementarity. [ABSTRACT FROM AUTHOR]

Herein, we detail several specific challenges that hinder the effective synthesis of glutamyl hydrazide containing peptides, and we describe a synthetic strategy to work around these challenges. Glutamyl hydrazide is an unnatural amino acid residue that bears an acyl hydrazide functional group on its side chain. This family of compounds has the potential to provide potent and selective inhibitor molecules for several families of enzymes. During peptide synthesis, however, these side chains—even in protected form—can derail the synthesis by initiating undesired side reactions. Avoiding these side reactions is critical for enabling effective access to this family of compounds. [ABSTRACT FROM AUTHOR]

Carbon–sulfur bond-formation reactions are applied widely in organic synthesis and chemical biology. Hydrothiolation of alkenes provides a direct way to build carbon–sulfur bonds. Most known methods proceed via radical processes and result in anti-Markovnikov-type products. Herein, we demonstrate that I2 catalyzes the hydrothiolation of alkenes and provides Markovnikov-type products in good to excellent yields. Titration studies revealed that thiol was activated by I2 through halogen bonding. This metal-free reaction has advantages such as green and mild conditions, high functionality tolerance, broad substrate scope, and atom economy. Its application was further demonstrated in peptide syntheses. [ABSTRACT FROM AUTHOR]

Class A serine β-lactamases (SBLs) have a conserved non-active site structural domain called the omega loop (Ω-loop), in which a glutamic acid residue is believed to be directly involved in the hydrolysis of β-lactam antibiotics by providing a water molecule during catalysis. We aimed to design and characterise potential pentapeptides to mask the function of the Ω-loop of β-lactamases and reduce their efficacy, along with potentiating the β-lactam antibiotics and eventually decreasing β-lactam resistance. Considering the Ω-loop sequence as a template, a group of pentapeptide models were designed, validated through docking, and synthesised using solid-phase peptide synthesis (SPPS). To check whether the β-lactamases (BLAs) were inhibited, we expressed specific BLAs (TEM-1 and SHV-14) and evaluated the trans-expression through a broth dilution method and an agar dilution method (HT-SPOTi). To further support our claim, we conducted a kinetic analysis of BLAs with the peptides and employed molecular dynamics (MD) simulations of peptides. The individual presence of six histidine-based peptides (TSHLH, ETHIH, ESRLH, ESHIH, ESRIH, and TYHLH) reduced β-lactam resistance in the strains harbouring BLAs. Subsequently, we found that the combinational effect of these peptides and β-lactams sensitised the bacteria towards the β-lactam drugs. We hypothesize that the antimicrobial peptides obtained might be considered among the novel inhibitors that can be used specifically against the Ω-loop of the β-lactamases. [ABSTRACT FROM AUTHOR]

3‐Amino‐2H‐azirines are potentially versatile building blocks in heterocyclic and peptide synthesis. Three new 3‐amino‐2H‐azirines have been synthesized as racemates or mixtures of diastereoisomers in cases where another chiral residue is incorporated as the exocyclic amine. The crystal structures of two of them, an approximately 1:1 diastereoisomeric mixture of (2R)‐ and (2S)‐2‐ethyl‐3‐[(2S)‐2‐(1‐methoxy‐1,1‐diphenylmethyl)pyrrolidin‐1‐yl]‐2‐methyl‐2H‐azirine, C23H28N2O, 11, and 2‐benzyl‐3‐(N‐methyl‐N‐phenylamino)‐2‐phenyl‐2H‐azirine, C22H20N2, 12, and the third as its diastereoisomeric trans‐PdCl2 complex, trans‐dichlorido[(2R)‐2‐ethyl‐2‐methyl‐3‐(X)‐2H‐azirine][(2S)‐2‐ethyl‐2‐methyl‐3‐(X)‐2H‐azirine]palladium(II), where X = N‐{[(1S,2S,5S)‐6,6‐dimethylbicyclo[3.1.1]heptan‐2‐yl]methyl}‐N‐phenylamino, [PdCl2(C21H30N2)2], 14, have been determined and the geometries of the azirine rings compared with those of 11 other 3‐amino‐2H‐azirine structures reported in the literature. Most notable is the very long formal N—C single bond, which is, with one exception, around 1.57 Å. Each compound has crystallized in a chiral space group. The Pd atom in the trans‐PdCl2 complex is coordinated by one of each of the pair of diastereoisomers, while both of the diastereoisomers share the same crystallographic site in the structure of 11; this property thereby manifesting itself as disorder. The chosen crystal of 12 is either an inversion twin or composed of a pure enantiomorph, but this could not be established specifically. [ABSTRACT FROM AUTHOR]

Two Streptomyces strains, named N11-26 and DC10-5, were isolated from deep-sea and non-photosynthetic stony coral, respectively. Strain N11-26 produces lobophorin C and divergolides, which are antimicrobial substances. This study aimed to classify these strains and reveal their cryptic potential to synthesize other secondary metabolites, such as polyketides and nonribosomal peptides. Strains N11-26 and DC10-5 showed 16S rRNA gene sequence similarities of 100% and 99.9% to Streptomyces olivaceus NRRL B-3009T, respectively. By digital DNA–DNA hybridization using whole-genome sequences, these strains were classified as Streptomyces olivaceus. Strain N11-26 was closer to the type strain of S. olivaceus than strain DC10-5 and possessed 17 clusters of polyketide synthase (PKS) and/or nonribosomal peptide synthetases (NRPS) genes, whereas strain DC10-5 harbored 19 clusters. Putative products by these gene clusters were predicted by bioinformatic analyses. Although 15 clusters were conserved between the two strains, two and four clusters were specific in strains N11-26 and DC10-5, respectively. This represents a diversity of potential polyketide and nonribosomal peptide compounds between strains of S. olivaceus. To the best of our knowledge, this is the first report annotating all the PKS and NRPS gene clusters in S. olivaceus strains with their putative products to provide useful information for genome mining. [ABSTRACT FROM AUTHOR]

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The venom toxin δ-ctenitoxin-Pn2a of the spider Phoneutria nigriventer can cause severe envenomation in humans. Furthermore, the cystine-knot motif of δ-ctenitoxin-Pn2a provides exceptional stability, thereby hampering immune response activation. Here we identified epitope G34YFWIAWYKLANCKK48 from δ-ctenitoxin-Pn2a through the Immune Epitope Database Analysis Resource and used it to design antigenic peptides. The Cys residue was replaced by α-aminobutyric acid (Abu) to prevent disulfide bond formation. To increase the immunogenicity of these molecules, branched and N-palmitoylated versions were synthesized. Ac-GYFWIAWYKLAN-Abu-KKG-NH2 (A), Palm-GYFWIAWYKLAN-Abu-KKG-NH2 (B) and (Ac-GYFWIAWYKLAN-Abu-KK)2-KG-NH2 (C) were prepared by solid-phase synthesis and their identity was confirmed by ESI–MS. They were then studied by RP-HPLC and all the chromatograms obtained showed only one main peak. Cytotoxicity was evaluated on the murine macrophage cell line RAW 264.7 using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay in the presence of increasing doses of each peptide (0.25–10.0 µM). Peptide A did not show cytotoxicity between 0.25 and 10.0 µM, while B and C did at concentrations equal or over 0.5 and 10.0 respectively. The cellular distribution of NF-κB was examined by immunofluorescence after exposing macrophages to 0.5 µM of each peptide. Early activation was observed for all three peptides, thereby indicating that they are promising immunogens for antivenom production. Nevertheless, in vivo tests are still required to assess their immunogenic capacity and whether the antibodies generated can confer protection against the venom. [ABSTRACT FROM AUTHOR]

The replacement of the α-carbon atom in an α-amino acid structure by a nitrogen atom yields alkylcarbazic acids, also known as α-aza amino acids. Although the topology of α-amino acids and α-aza amino acids is similar, their chemical and stereochemical properties are significantly different. For this reason, the application of the common solid-phase peptide synthesis (SPPS) protocol cannot be used for aza-peptide bond synthesis without changes. On the other hand, the aza-peptide bond is more stable than the common peptide bond, therefore these compounds are very attractive targets for drug design. In this review, we summarize data on aza-peptide bond chemistry, with implications for the improvement of aza-peptide chemical synthesis.

Bacicyclin is a cyclic hexapeptide with antibacterial activity against Enterococcus faecalis and Staphylococcus aureus with minimum inhibition concentration (MIC) values of 8 and 12 µM, respectively. Studies on a reversed sequence of bacicyclin were conducted to investigate how the reversed peptide sequence affects its biological properties. A reversed-bacicylin, cyclo-(Gly-Leu-Val-Ile-Ala-Phe), was successfully synthesized by constructing the linear precursor on 2-chlorotrityl chloride resin using a Fmoc-based strategy. The HATU/HOAt reagent was applied in all peptidic bond formations, and the desired linear hexapeptide (82% yield) was cleaved off the resin using a mixture of trifluoroacetic acid:dichloromethane (2:8). The linear peptide was cyclized using 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium-3-oxidehexa-fluorophosphate (HATU) as a coupling agent and diisopropylethylamine (DIPEA) as the base in a very dilute solution (0.001 M) in dichloromethane, then purified by octadecyl silica gel (ODS) column chromatography to obtain the reversed-bacicyclin (43.7% yield). The purity of the cyclic product was analyzed using analytical RP-HPLC (tR = 20.01 min), and characterized by HR-TOF-MS, 1H-NMR, and 13C-NMR. The biological activity of the reversed-bacicyclin is much lower compared to bacicyclin, indicating that the amino acid sequence of the cyclopeptide dictates the antibacterial activity. This finding gives additional information on the relationship between peptide sequence and biological properties.