Leveraging the Mukaiyama oxidation-reduction condensation reaction for on-resin aryl thio-esterification for bio-conjugation.

A room-temperature Mukaiyama oxidation-reduction condensation inspired thioesterification methodology has been developed to afford aryl Cα-terminal peptide thioesters on-resin. The conditions herein feature mild reactions compatible with all Fmoc-SPPS protocols offering direct access to this critical arylthioester scaffold. This one-pot synthesis to aryl-thioester functionalised peptides facilitates peptide/protein synthesis by native chemical ligation.

Synthesis, Structure-Activity Relationship Study, Bioactivity, and Nephrotoxicity Evaluation of the Proposed Structure of the Cyclic Lipodepsipeptide Brevicidine B.

The brevicidines represent a novel class of nonribosomal antimicrobial peptides that possess remarkable potency and selectivity toward highly problematic and resistant Gram-negative pathogenic bacteria. A recently discovered member of the brevicidine family, coined brevicidine B (2), comprises a single amino acid substitution (from d-Tyr2 to d-Phe2) in the amino acid sequence of the linear moiety of brevicidine (1) and was reported to exhibit broader antimicrobial activity against both Gram-negative (MIC = 2-4 µgmL-1) and Gram-positive (MIC = 2-8 µgmL-1) pathogens. Encouraged by this, we herein report the first total synthesis of the proposed structure of brevicidine B (2), building on our previously reported synthetic strategy to access brevicidine (1). In agreement with the original isolation paper, pleasingly, synthetic 2 demonstrated antimicrobial activity toward Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae (MIC = 4-8 µgmL-1). Interestingly, however, synthetic 2 was inactive toward all of the tested Gram-positive pathogens, including methicillin-resistant Staphylococcus aureus strains. Substitution of d-Phe2 with its enantiomer, and other hydrophobic residues, yields analogues that were either inactive or only exhibited activity toward Gram-negative strains. The striking difference in the biological activity of our synthetic 2 compared to the reported natural compound warrants the re-evaluation of the original natural product for purity or possible differences in relative configuration. Finally, the evaluation of synthetic 1 and 2 in a human kidney organoid model of nephrotoxicity revealed substantial toxicity of both compounds, although 1 was less toxic than 2 and polymyxin B. These results indicate that modification to position 2 may afford a strategy to mitigate the nephrotoxicity of brevicidine.

NMR Shows Why a Small Chemical Change Almost Abolishes the Antimicrobial Activity of Glycocin F.

Glycocin F (GccF), a ribosomally synthesized, post-translationally modified peptide secreted by Lactobacillus plantarum KW30, rapidly inhibits the growth of susceptible bacteria at nanomolar concentrations. Previous studies have highlighted structural features important for its activity and have shown the absolute requirement for the Ser18 O-linked GlcNAc on the eight-residue loop linking the two short helices of the (C-X6-C)2 structure. Here, we show that an ostensibly very small chemical modification to Ser18, the substitution of the Cα proton with a methyl group, reduces the antimicrobial activity of GccF 1000-fold (IC50 1.5 µM cf. 1.5 nM). A comparison of the GccFα-methylSer18 NMR structure (PDB 8DFZ) with that of the native protein (PDB 2KUY) showed a marked difference in the orientation and mobility of the loop, as well as a markedly different positioning of the GlcNAc, suggesting that loop conformation, dynamics, and glycan presentation play an important role in the interaction of GccF with as yet unknown but essential physiological target molecules.

On-Resin Conjugation of the Ruthenium Anticancer Agent Plecstatin-1 to Peptide Vectors.

Ruthenium piano-stool complexes have been explored for their anticancer activity and some promising compounds have been reported. Herein, we conjugated a derivative of plecstatin-1 to peptides in order to increase their cancer cell targeting ability. For this purpose, plecstatin-1 was modified at the arene ligand to introduce a functional amine handle (3), which resulted in a compound that showed similar activity in an in vitro anticancer activity assay. The cell-penetrating peptide TAT48-60, tumor-targeting neurotensin8-13, and plectin-targeting peptide were functionalized with succinyl or ß-Ala-succinyl linkers under standard solid-phase peptide synthesis (SPPS) conditions to spatially separate the peptide backbones from the bioactive metal complexes. These modifications allowed for conjugating precursor 3 to the peptides on resin yielding the desired metal-peptide conjugates (MPCs), as confirmed by high-performance liquid chromatography (HPLC), NMR spectroscopy, and mass spectrometry (MS). The MPCs were studied for their behavior in aqueous solution and under acidic conditions and resembled that of the parent compound plecstatin-1. In in vitro anticancer activity studies in a small panel of cancer cell lines, the TAT-based MPCs showed the highest activity, while the other MPCs were virtually inactive. However, the MPCs were significantly less active than the small molecules plecstatin-1 and 3, which can be explained by the reduced cell uptake as determined by inductively coupled plasma MS (ICP-MS). Although the MPCs did not display potent anticancer activities, the developed conjugation strategy can be extended toward other metal complexes, which may be able to utilize the targeting properties of peptides.

Factors influencing on-resin depsipeptide bond formation: case studies on daptomycin- and brevicidine-derived sequences.

Depsipeptides are an important class of bioactive natural products, where a growing number of genome-mined structures that display anti-microbial activity are macrocyclic depsipeptides. Chemically, peptide ester (depsipeptide) bond formation often displays low yields, and thereby hampers efforts to access these structures for structure-activity studies. Herein, we present a systematic study of the variables that influence depsipeptide bond formation on-resin, using simplified sequences derived from antibiotic peptides, daptomycin and brevicidine, prepared via Fmoc-based solid-phase synthesis. Our study highlights reaction solvent as the key determinant, where switching the solvent from DMF to DCM in almost all cases increased the amount of depsipeptide product. Limiting the number of amino-acids N-terminal to the reactive alcohol was also noted to significantly improve the acylation efficiency. The impact of different N-terminal and side-chain protecting groups, as well as stereochemistry, was also investigated. Additives to the reaction, such as inclusion of surfactants for esterification of long hydrophobic sequences, did not improve conversion. 6-ClHOBt, often added to improve acylation efficiency, notably decreased the amount of depsipeptide observed. Lastly, no significant difference between polystyrene and Tentagel® (PEG-decorated) resins were found for these sequences.

Harnessing the power of a photoinitiated thiol-ene "click" reaction for the efficient synthesis of S-lipidated collagen model peptide amphiphiles.

A photoinitiated thiol-ene "click" reaction was used to synthesize S-lipidated collagen model peptide amphiphiles. Use of 2-iminothiolane provided an epimerization-free thiol handle required for thiol-ene based incorporation of lipid moieties onto collagen-based peptide sequences. This approach not only led to improvements in the triple helical characteristics of the resulting collagen model peptides but also increased the aqueous solubility of the peptide amphiphiles. As a result, this methodology holds significant potential for the design and advancement of functional peptide amphiphiles, offering enhanced capabilities across a wide range of applications.

Flow-Based Fmoc-SPPS Preparation and SAR Study of Cathelicidin-PY Reveals Selective Antimicrobial Activity.

Antimicrobial peptides (AMPs) hold promise as novel therapeutics in the fight against multi-drug-resistant pathogens. Cathelicidin-PY (NH2-RKCNFLCKLKEKLRTVITSHIDKVLRPQG-COOH) is a 29-residue disulfide-cyclised antimicrobial peptide secreted as an innate host defence mechanism by the frog Paa yunnanensis (PY) and reported to possess broad-spectrum antibacterial and antifungal properties, exhibiting low cytotoxic and low hemolytic activity. Herein, we detail the total synthesis of cathelicidin-PY using an entirely on-resin synthesis, including assembly of the linear sequence by rapid flow Fmoc-SPPS and iodine-mediated disulfide bridge formation. By optimising a synthetic strategy to prepare cathelicidin-PY, this strategy was subsequently adapted to prepare a bicyclic head-to-tail cyclised derivative of cathelicidin-PY. The structure-activity relationship (SAR) of cathelicidin-PY with respect to the N-terminally positioned disulfide was further probed by preparing an alanine-substituted linear analogue and a series of lactam-bridged peptidomimetics implementing side chain to side chain cyclisation. The analogues were investigated for antimicrobial activity, secondary structure by circular dichroism (CD), and stability in human serum. Surprisingly, the disulfide bridge emerged as non-essential to antimicrobial activity and secondary structure but was amenable to synthetic modification. Furthermore, the synthetic AMP and multiple analogues demonstrated selective activity towards Gram-negative pathogen E. coli in physiologically relevant concentrations of divalent cations.

N-Vinyl Acrylamides: Versatile Heterobifunctional Electrophiles for Thiol-Thiol Bioconjugations.

Herein we report the first examples of thiol-selective heterobifunctional electrophiles, N-vinyl acrylamides, that enable efficient highly selective thiol-thiol bioconjugations and cysteine modification of peptides. We demonstrate that these new classes of thiol-selective scaffolds can readily undergo a thia-Michael addition and an orthogonal radical induced thiol-ene "click" reaction under biocompatible conditions. Furthermore, the formation of an unexpected Markovnikov N,S-acetal hydrothiolation was explained using computational studies. We also reveal that N-methylation of the N-vinyl acrylamide scaffold changes the regioselectivity of the reaction. We demonstrate that use of N-vinyl acrylamides shows promise as an efficient, mild, and exquisite cysteine-selective protocol for facile construction of fluorophore-labeled peptides and proteins and that the resultant conjugates are resistant to degradation and thiol exchange, thus significantly improving their biophysical properties.

Discovery, Synthesis, and Optimization of Peptide-Based Antibiotics.

The rise of multidrug resistant bacteria has significantly compromised our supply of antibiotics and poses an alarming medical and economic threat to society. To combat this problem, it is imperative that new antibiotics and treatment modalities be developed, especially those toward which bacteria are less capable of developing resistance. Peptide natural products stand as promising candidates to meet this need as bacterial resistance is typically slow in response to their unique modes of action. They also have additional benefits including favorable modulation of host immune responses and often possess broad-spectrum activity against notoriously treatment resistant bacterial biofilms. Moreover, nature has provided a wealth of peptide-based natural products from a range of sources, including bacteria and fungi, which can be hijacked in order to combat more dangerous clinically relevant infections.This Account highlights recent advances in the total synthesis and development of a range of peptide-based natural product antibiotics and details the medicinal chemistry approaches used to optimize their activity.In the context of antibiotics with potential to treat Gram-positive bacterial infections, this Account covers the synthesis and optimization of the natural products daptomycin, glycocin F, and alamethicin. In particular, the reported synthesis of daptomycin highlights the utility of on-resin ozonolysis for accessing a key kynurenine residue from the canonical amino acid tryptophan. Furthermore, the investigation into glycocin F analogues uncovered a potent lead compound against Lactobacillus plantarum that bears a non-native thioacetal linkage to a N-acetyl-d-glucosamine (GlcNAc) sugar, which is otherwise O-linked in its native form.For mycobacterial infections, this Account covers the synthesis and optimization of teixobactin, callyaerin A, lassomycin, and trichoderin A. The synthesis of callyaerin A, in particular, highlighted the importance of a (Z)-2,3-diaminoacrylamide motif for antimicrobial activity against Mycobacterium tuberculosis, while the synthesis of trichoderin A highlighted the importance of (R)-stereoconfiguration in a key 2-amino-6-hydroxy-4-methyl-8-oxodecanoic acid (AHMOD) residue.Lastly, this Account covers lipopeptide antibiotics bearing activity toward Gram-negative bacterial infections, namely, battacin and paenipeptin C. In both cases, optimization of the N-terminal lipid tails led to the identification of analogues with potent activity toward Escherichia coli and Pseudomonas aeruginosa.

A Solid Support-Based Synthetic Strategy for the Site-Selective Functionalization of Peptides with Organometallic Half-Sandwich Moieties.

The number of donor atoms available on peptides that can competitively coordinate to metal centers renders the site-selective generation of advanced metal-peptide conjugates in high purity a challenging venture. Herein, we present a transmetalation-based synthetic approach on solid support in which an imidazolium pro-ligand can be used to selectively anchor a range of transition metal half-sandwich complexes onto peptides in the presence of multiple coordinative motifs. Amenable to solid support, a range of N-terminus and/or lysine conjugated metal-peptide conjugates were obtained in high purity after cleavage from the resin. The metalated peptides were evaluated for their anticancer properties against human cancer cell lines. While no cytotoxic activity was observed, this platform has the potential to i) provide a pathway to site-selective peptide labelling, ii) be explored as a biorthogonal handle and/or iii) generate a new strategy for ligand design in transition metal catalysts.

Synthetic Studies towards the Calcium-Dependent Lipopeptide Antibiotic Cadaside B.

In the current global crisis of antimicrobial resistance, antimicrobial peptides represent a promising source of alternative antibiotics. Recently discovered cadaside B, a novel calcium-dependent antibiotic, exhibits potent antimicrobial activity towards Gram-positive pathogens including multi-drug resistant strains. These properties, coupled with a novel structure, non-cytotoxicity, and low likelihood of developing resistance render cadaside B an important synthetic target. Herein, a synthetic strategy towards cadaside B is reported with the key steps involving on-resin depsipeptide bond formation and solution-phase macrolactamization. Good agreement of the synthetic cadaside B MS/MS fragmentation pattern was observed with the natural product, but a different 1 H NMR spectrum and absence of antimicrobial activity suggest an undetected epimerization event took place during the synthesis. Herein the findings of our synthetic journey and suggestions for future directions are presented.

Photo-induced radical thiol-ene chemistry: a versatile toolbox for peptide-based drug design.

While the global market for peptide/protein-based therapeutics is witnessing significant growth, the development of peptide drugs remains challenging due to their low oral bioavailability, poor membrane permeability, and reduced metabolic stability. However, a toolbox of chemical approaches has been explored for peptide modification to overcome these obstacles. In recent years, there has been a revival of interest in photoinduced radical thiol-ene chemistry as a powerful tool for the construction of therapeutic peptides.

PAC1, VPAC1, and VPAC2 Receptor Expression in Rat and Human Trigeminal Ganglia: Characterization of PACAP-Responsive Receptor Antibodies.

Pituitary adenylate cyclase-activating peptide (PACAP) is a neuropeptide expressed in the trigeminal ganglia (TG). The TG conducts nociceptive signals in the head and may play roles in migraine. PACAP infusion provokes headaches in healthy individuals and migraine-like attacks in patients; however, it is not clear whether targeting this system could be therapeutically efficacious. To effectively target the PACAP system, an understanding of PACAP receptor distribution is required. Therefore, this study aimed to characterize commercially available antibodies and use these to detect PACAP-responsive receptors in the TG. Antibodies were initially validated in receptor transfected cell models and then used to explore receptor expression in rat and human TG. Antibodies were identified that could detect PACAP-responsive receptors, including the first antibody to differentiate between the PAC1n and PAC1s receptor splice variants. PAC1, VPAC1, and VPAC2 receptor-like immunoreactivity were observed in subpopulations of both neuronal and glial-like cells in the TG. In this study, PAC1, VPAC1, and VPAC2 receptors were detected in the TG, suggesting they are all potential targets to treat migraine. These antibodies may be useful tools to help elucidate PACAP-responsive receptor expression in tissues. However, most antibodies exhibited limitations, requiring the use of multiple methodologies and the careful inclusion of controls.

Characterization of Antibodies against Receptor Activity-Modifying Protein 1 (RAMP1): A Cautionary Tale.

Calcitonin gene-related peptide (CGRP) is a key component of migraine pathophysiology, yielding effective migraine therapeutics. CGRP receptors contain a core accessory protein subunit: receptor activity-modifying protein 1 (RAMP1). Understanding of RAMP1 expression is incomplete, partly due to the challenges in identifying specific and validated antibody tools. We profiled antibodies for immunodetection of RAMP1 using Western blotting, immunocytochemistry and immunohistochemistry, including using RAMP1 knockout mouse tissue. Most antibodies could detect RAMP1 in Western blotting and immunocytochemistry using transfected cells. Two antibodies (844, ab256575) could detect a RAMP1-like band in Western blots of rodent brain but not RAMP1 knockout mice. However, cross-reactivity with other proteins was evident for all antibodies. This cross-reactivity prevented clear conclusions about RAMP1 anatomical localization, as each antibody detected a distinct pattern of immunoreactivity in rodent brain. We cannot confidently attribute immunoreactivity produced by RAMP1 antibodies (including 844) to the presence of RAMP1 protein in immunohistochemical applications in brain tissue. RAMP1 expression in brain and other tissues therefore needs to be revisited using RAMP1 antibodies that have been comprehensively validated using multiple strategies to establish multiple lines of convincing evidence. As RAMP1 is important for other GPCR/ligand pairings, our results have broader significance beyond the CGRP field.

Synthesis and Systematic Study on the Effect of Different PEG Units on Stability of PEGylated, Integrin-αvß6-Specific A20FMDV2 Analogues in Rat Serum and Human Plasma.

A20FMDV2 is a 20-mer peptide that exhibits high selectivity and affinity for the tumour-related αvß6 integrin that can compete with extracellular ligands for the crucial RGD binding site, playing a role as a promising αvß6-specific inhibitor for anti-cancer therapies. Unfortunately, the clinical value of A20FMDV2 is limited by its poor half-life in blood caused by rapid renal excretion and its reported high susceptibility to serum proteases. The incorporation of poly (ethylene glycol) chains, coined PEGylation, is a well-established approach to improve the pharmacokinetic properties of drug molecules. Here, we report a systematic study on the incorporation of a varying number of ethylene glycol units (1-20) into the A20FMDV2 peptide to establish the effects of PEGylation size on the peptide stability in both rat serum and human plasma. In addition, the effect of acetyl and propionyl PEGylation handles on peptide stability is also described. Selected peptide analogues were assessed for integrin-αvß6-targeted binding, showing good specificity and activity in vitro. Stability studies in rat serum established that all of the PEGylated peptides displayed good stability, and an A20FMDV2 peptide containing twenty ethylene glycol units (PEG20) was the most stable. Surprisingly, the stability testing in human plasma identified shorter PEGs (PEG2 and PEG5) as more resistant to degradation than longer PEGs, a trend which was also observed with affinity binding to integrin αvß6.

Thiol-ene enabled preparation of S-lipidated anti-HBV peptides.

Despite significant efforts made towards treatments for Hepatitis B virus (HBV), a long-term curative treatment has thus far eluded scientists. Recently, the Sodium Taurocholate Co-Transporting Polypeptide (NTCP) receptor has been identified as the entry pathway of HBV into hepatocytes. Myrcludex B, an N-terminally myristoylated 47-mer peptide mimic of the preS1 domain of the Hepatitis B virion, was identified as a potent protein-protein interaction (PPI) inhibitor blocking HBV fusion (IC50 = 140 pM). Herein we report an optimised chemical synthesis of Myrcludex B and a series of novel analogues. Employing a small modification to the Cysteine Lipidation of a Peptide or Amino acid (CLipPA) thiol-ene reaction, a library of S-lipidated Myrcludex B and truncated (21-mer) analogues were prepared, providing novel chemical space to probe for the discovery of novel anti-HBV peptides. The S-lipidated analogues showed an equivalent or a slight decrease (∼2-fold) in binding effectiveness to NTCP expressing hepatocytes compared to Myrcludex B. Three S-lipidated analogues were highly potent HBV inhibitors (IC50 0.97-3.32 nM). These results demonstrate that incorporation of heteroatoms into the lipid 'anchor' is tolerated by this antiviral scaffold and to the best of our knowledge constitutes the first report of potent S-lipidated antiviral peptides. Interestingly, despite only moderate reductions in binding effectiveness, truncated analogues possessed dramatically reduced inhibitory activity thus providing new insights into the structure activity relationship of these hitherto unreported antiviral S-lipopeptides.

The Total Chemical Synthesis and Biological Evaluation of the Cationic Antimicrobial Peptides, Laterocidine and Brevicidine.

Antimicrobial resistance is a significant threat to public health systems worldwide, prompting immediate attention to develop new therapeutic agents with novel mechanisms of action. Recently, two new cationic non-ribosomal peptides (CNRPs), laterocidine and brevicidine, were discovered from Brevibacillus laterosporus through a global genome-mining approach. Both laterocidine and brevicidine exhibit potent antimicrobial activity toward Gram-negative bacteria, including difficult-to-treat Pseudonomas aeruginosa and colistin-resistant Escherichia coli, and a low risk of resistance development. Herein, we report the first total syntheses of laterocidine and brevicidine via an efficient and high-yielding combination of solid-phase synthesis and solution-phase macrolactamization. The crucial depsipeptide bond of the macrolactone rings of laterocidine and brevicidine was established on-resin between the side-chain hydroxy group of Thr9 with Alloc-Gly-OH or Alloc-Ser(tBu)-OH, respectively. A conserved glycine residue within the lactone macrocycle is exploited for the initial immobilization onto the hyper acid-labile 2-chlorotrityl chloride resin, subsequently enabling an efficient solution-phase macrocyclization to yield laterocidine and brevicidine in 36% and 10% overall yields, respectively (with respect to resin loading). A biological evaluation against both Gram-positive and Gram-negative bacteria demonstrated that synthetic laterocidine and brevicidine possessed a potent and selective antimicrobial activity toward Gram-negative bacteria, in accordance with the isolated compounds.

An Insight into FDA Approved Antibody-Drug Conjugates for Cancer Therapy.

The large number of emerging antibody-drug conjugates (ADCs) for cancer therapy has resulted in a significant market 'boom', garnering worldwide attention. Despite ADCs presenting huge challenges to researchers, particularly regarding the identification of a suitable combination of antibody, linker, and payload, as of September 2021, 11 ADCs have been granted FDA approval, with eight of these approved since 2017 alone. Optimism for this therapeutic approach is clear, despite the COVID-19 pandemic, 2020 was a landmark year for deals and partnerships in the ADC arena, suggesting that there remains significant interest from Big Pharma. Herein we review the enthusiasm for ADCs by focusing on the features of those approved by the FDA, and offer some thoughts as to where the field is headed.

Recent Progress in the Synthesis of Homogeneous Erythropoietin (EPO) Glycoforms.

Erythropoietin (EPO) has been regarded as a therapeutic glycoprotein for the clinical treatment of anaemia since its approval by the Food and Drug Administration (FDA) in 1989. Commercial production of the 165-residue glycoprotein is by recombinant protein expression using mammalian cell lines that renders a complex mixture of glycoforms that have an identical amino acid sequence but variations in the structures of the pendant glycans. This heterogeneous nature of human recombinant EPO restricts structural and bioactivity studies in medicinal chemistry. Consequently, chemical synthesis provides an elegant approach for the preparation of complex homogeneous glycoproteins from a readily accessible pool of amino acids and sugars. In addition, the combination of chemical and biosynthesis enables robust and large-scale production of homogeneous EPO. The scope of this minireview is to summarise the recent advances in the chemical and semisyntheses of homogeneous EPO glycoforms, highlighting the versatile approaches to the preparation and structural manipulations of the carbohydrate chains incorporated into synthetic EPO glycoproteins.

Variable-Length Ester-Based Staples for α-Helical Peptides by Using A Double Thiol-ene Reaction.

A novel peptide stapling method effected by a double thiol-ene reaction between two cysteine residues and a divinyl diester to access stapled peptides with enhanced cell permeability is reported. This diverse chemical tool kit provides facile access to stapled peptides with varying bridge lengths. Stapled Axin mimetics were synthesised by using this stapling method resulting in improved α-helicity relative to the unstapled peptide. Cell penetrating stapled analogues of the SIGK peptide that targets the protein-protein interaction hotspot of Gßγ proteins were also synthesised that exhibited a moderate increase in α-helicity and were cell permeable. This chemoselective peptide stapling method is highly amenable as a facile method to easily modify synthetic α-helical peptides to target intracellular proteins.