The dramatic increase in antimicrobial resistance has led to active research for new treatments. Antimicrobial peptides (AMPs) are small 7 to 100 amino acids [1, 2]. One discovered mode of action of AMPs is membrane disruptive activity, associated with the interaction with membrane phospholipids [3]. Another way of action of AMPs is intracellular targeting. Some AMPs can cross the cell barriers and reach targets in the cytoplasm [2]. For some AMPs, the occurrence of bivalent metal ions (such as Zn2+ and Cu2+) affects their activity or mode of action either because of binding metal ions, so that microbes cannot get enough nutrients essential for their life and virulence [4] or because AMPs need the metal ion as a booster of their antimicrobial activity, affecting the charge or the structure of the peptide [5]. Here, we discuss the action mechanism of Holothuroidin I (HLGHHALDHLLK) [6], a natural derived peptide from Mediterranean Sea cucumber Holothuria tubulosa, of the interactions with important metal ions located in mammals (such as Zn2+ and Cu2+).
Zinc (II), is the second most abundant transition metal in living organisms, [7] and present in superoxide dismutases, central enzymes in bacteria and fungi, associated with the detoxification of ROS generated by host cells during host-pathogen interactions [8]. Copper is another essential metal ion, which can switch between oxidized (Cu2+) and reduced (Cu+) states. It is essential for enzymes involved in cellular respiration, iron transport, and superoxide dismutation. Besides, the redox activity of copper may also contribute to its toxicity to microbes (and mammals) through classic copper-catalyzed Fenton chemistry and an increase in reactive oxygen species [9].
The action mechanism evaluation was performed using potentiometric titrations, conductivity studies, UV-Vis studies, voltammetric methods, and density function theory studies, giving valuable inside into the mechanism of interactions of Holothuroidin I with copper and zinc.

REFERENCES
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[2] F. H. Waghu, R. S. Barai, P. Gurung and S. Idicula-Thomas, Nucleic Acids Res., 2016, 44, D1094–D1097.
[3] J. D. F. Hale and R. E. W. Hancock, Expert Rev. Anti-Infect. Ther., 2007, 5 (6), 951–959.
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[5] A. Som, L. Yang, G. C. L. Wong and G. N. Tew, J. Am. Chem. Soc., 2009, 131 (42), 15102–15103.
[6] Domenico Schillaci, Maria Grazia Cusimano, Vincenzo Cunsolo, Rosaria Saletti, Debora Russo, Mirella Vazzana, Maria Vitale and Vincenzo Arizza, AMB Express 2013, 3:35.
[7] M. Vasak and D. W. Hasler, Curr. Opin. Chem. Biol., 2000, 4, 177–183.
[8] C. S. Hwang, G. Rhie, J. H. Oh, W. K. Huh, H. S. Yim and S. O. Kang, Microbiology, 2002, 148, 3705–3713.
[9] J. A. Lemire, J. J. Harrison and R. J. Turner, Nat. Rev. Microbiol., 2013, 11(6), 371–381.

Protein farnesylation is a post-translational modification where a 15 carbon farnesyl isoprenoid is appended to the C-terminal end of a protein by Farnesyltransferase (FTase). In the canonical understanding of FTase, the isoprenoids are attached to the Cysteine residue of a four amino acid CaaX box sequence. However, recent work has shown that five amino acid sequences can be recognized, such as the pentapeptide CMIIM. This new discovery greatly increases the number of potential FTase substrates, as the FTase is already known to tolerate a wide variety of amino acids in the canonical CaaX sequence. Due to the large number of potential substrates it is difficult to assay for novel CaaX sequences. With the goal of developing a more rapid and methodical method to evaluate potential substrates, we envisioned using MALDI to assay libraries of 10 peptides at a time, varying one amino acid in the CaaX box to all 20 canonical amino acids over two libraries. Through this method we observed 30 hits in the mass spectrum and chose eight for further evaluation. Seven of these sequences are novel substrates for FTase, with several meeting or surpassing the in vitro efficiency of the benchmark sequence CMIIM. Additionally, in vivo experiments in yeast demonstrate that proteins bearing these sequences can be efficiently prenylated in a biological context.

Peptide secondary structures have privileged roles in molecular recognition and therapeutic potential. α-Amino lactam residues have been commonly used as conformational constraints to study peptide structure-activity relationship for drug discovery [1]. N-Aminoimidazolone (Nai) residues offer similar means for constraining peptide backbone geometry [1, 2]. In model peptides, (4-methyl)Nai residues were found to adopt the central position of β- and γ-turn secondary structures. The addition of substituents at the 4- and 5-positions of the Nai residues may be used to mimic side chain function and orientation [3, 4]. Our presentation will feature the synthesis and application of Nai residues in the study of peptide structure-activity relationships [5].

1. St-Cyr D.J., García-Ramos Y., Doan N.D., Lubell W.D. Peptidomimetics I. Springer; Cham, Switzerland: 2017. Aminolactam, N-Aminoimidazolone, and N-Aminoimdazolidinone Peptide Mimics; pp. 125–175.
2. Proulx, C.; Lubell, W. D. "N-Amino-imidazolin-2-one Peptide Mimic Synthesis and Conformational Analysis" Lett., 2012, 14, 4552.
3. Poupart, J., Doan-Ngoc, D., Bérubé, D., Hamdane, Y., Medena, C., Lubell, W. D. "Palladium-Catalyzed Arylation of N-Aminoimidazol-2-ones towards Synthesis of Constrained Phenylalanine Dipeptide Mimics" Heterocycles, 2019, 99, 279-293
4. Poupart, J.; Hamdane, Y.; Lubell, W.D. "Synthesis of enantiomerically enriched 4,5-disubstituted N-aminoimidazol-2-one (Nai) peptide turn mimics" J. Chem. 2020, 98, 278–284.
5. Hamdane, Y.; Chauhan, S. P.; Vutla, S.; Mulumba, D.; Ong, H.; Lubell, W. D. Submitted.

Peptides are promising tools for designing sensitive and stable biosensors. For example, the redox self-assembled monolayer (SAM) based on the sequence Fc-Glu-(Ala)₂-Cys-NH₂ was successful evaluated as transducing interface in electrochemical biosensors. The design of such peptides includes: 1) cysteine to bind covalently the peptide to the gold electrode; 2) glutamic acid in N-terminal position to bind the ferrocene (Fc) in the amine group, and the antibody in the δ-carboxyl group, and 3) alanine to form a hydrophobic layer. Herein, we present the solid-phase synthesis of three different peptides with structure Fc-Glu-(X)₂-Cys-NH₂ (X=Ser, Gly or Phe) and the electrochemical behavior of the obtained SAMs. The Gly was chosen because of its smallest side chain, while Ser and Phe present hydroxyl groups (for H-bonds) and aromatic (for π-π interaction), respectively. The synthesis successful of the HPLC purified peptides were confirmed by mass spectroscopy. From cyclic voltammetry and impedance-derived electrochemical capacitance spectroscopy results, all peptides present reversible redox processes, and electron transfer rates (k_ET ) ranging from 17 to 31 s^-1. Since the peptide with Gly residues presented both the highest surface coverage (Γ = 2.6x10^-10 mol/cm²) and electrochemical capacitance (C_µ = 270 µF/cm²) values, it can be potentially applied for biosensors designing.

The heavy chalcogen tellurium (⁵²Te) is a versatile element with many potential applications in chemical biology and biochemistry, including mass cytometry, fluorescence imaging, and protein structure determination. Using L-tellurienylalanine (TePhe), a mimic of the natural amino acid L-phenylalanine (Phe) in which the phenyl side chain is replaced by a nearly isosteric tellurophene ring, tellurium can be covalently incorporated into the proteome of prokaryotes and eukaryotes by endogenous translation machinery. Our goal is to generate proteins with near stoichiometric levels of Phe to TePhe substitutions, verify preservation of protein structure and activity upon TePhe incorporation, and ultimately exploit the site-specific tellurium centres as handles for crystallographic phasing, protein NMR spectroscopy, and bio-orthogonal reactivity.

Here we report conditions for the expression of TePhe containing proteins in a standard E. coli expression system and validate the ability of TePhe to act as an effective Phe analogue within a folded protein. Our target for TePhe incorporation is the streptococcal immunoglobulin-binding Protein G B1 domain (GB1), a remarkably heat-stable 56-residue domain containing 2 Phe residues which pack against one another within the domain’s hydrophobic core. In Phe-deficient media containing glyphosate as an inhibitor of aromatic amino acid biosynthesis, we obtained a GB1 mixture in which approximately 1 in 2 Phe sites were substituted by TePhe. Fractionation by reverse-phase HPLC allowed us to obtain a sample with 85% TePhe substitution as evidenced by amino acid analysis. Using ¹H-¹⁵N HSQC and circular dichroism spectroscopy, we find that TePhe effectively takes on the role of Phe, and alters the melting temperature of the protein by less than 5 °C.

Genetically encoded macrocyclic peptide libraries with unnatural pharmacophores are valuable sources for the discovery of ligands for many targets of interest. Traditionally, the generation of such libraries employs “early-stage” incorporation of unnatural building blocks into the chemically or translationally produced macrocycles. Here, we describe a divergent late-stage approach to such libraries starting from readily available starting material: genetically encoded libraries of peptides. A diketone linchpin 1,5-dichloropentane-2,4-dione converts peptide libraries displayed on phage to 1,3-diketone bearing macrocyclic peptides (DKMP): shelf-stable precursors for Knorr pyrazole synthesis. Ligation of diverse hydrazine derivatives onto DKMP libraries displayed on phage that carries silent DNA-barcodes yields macrocyclic libraries in which the amino acid sequence and the pharmacophore are encoded by DNA. The "late" nature of the 1,3-diketone and hydrazine ligation reaction makes it a favorable approach to graft irreversible and reversible covalent ligands, as well as other chemical fragments with minor modifications. Selection of this library against therapeutically important protein targets can enrich macrocycles containing fragments that target the protein of interest. The macrocycle can help to form favorable interactions with the protein target and increase the binding affinity when compared to the ligand or the pharmacophore alone. This methodology can graft diverse pharmacophores into many existing genetically encoded phage libraries and significantly increase the value of such libraries and aid in molecular discoveries for challenging protein targets.

Polyfluoroaromatic reagents readily react with thiols via S_NAr and provide excellent templates on to which peptides can be assembled through side-chain macrocyclisation.

We have previously investigated the reactivity of hexafluorobenzene (HFB) towards cysteine and related nucleophiles and we have subsequently extended these studies to fluorinated porphyrins, exploiting them as photoactive scaffolds for peptide cyclisation. We will describe how the di-substitution of tetrakis(pentafluorophenyl)porphyrin with the Skin Penetrating And Cell Entering (SPACE) peptide (ACTGSTQHQCG), presenting two cysteines in position i, i+8, successfully afforded the macrocyclised product under peptide-compatible conditions. By repeating this reaction with different peptide sequences and inter-thiol distances, we evaluate whether this chemistry is sequence-specific and we give important insights regarding the geometrical constraints of this macorcyclisation (e.g. minimal number of amino acids required to reach the para positions of adjacent meso phenyl groups). Moreover, crosslinking cysteines with tetrakis(pentafluorophenyl)porphyrin does not affect SPACE peptide uptake in skin cancer cells and the presence of the porphyrin core potentially imparts photodynamic properties to the conjugate. We anticipate that the remaining para positions are available for further conjugation and a similar methodology might also be applicable to bis(pentafluorophenyl)porphyrin.

Since a growing number of APIs are protein or peptide-based, we believe that this work, together with future studies, could expand the application of peptides as therapeutic and diagnostic agents.

The protocol to purify and fluorescently label proteins typically includes lengthy purification steps and stochastically governed labelling methods. When multi-labelled proteins are required, poor yields, waste of valuable reagents, and lengthy sample preparation times are generally inevitable. In addition, many fluorophore combinations are incompatible, which makes the task of efficiently producing multi-labelled, high-purity proteins very difficult.
Here, we describe a novel method that enables the preparation of purified and labelled proteins in only a few hours. Our protocol gives high yield of the desired protein product using minimal mutations, high specificity of labelling, and ease of chemically orthogonal attachment of other fluorescent probes in later steps. This technique takes advantage of native chemical ligation in a single-step, in one liquid-chromatography column, by the addition of one reagent, with the ability to purify and label any protein using any desired fluorescent probe.
We demonstrate the efficacy of this method on the eukaryotic initiation factor 4E binding partner 2 (4E-BP2). This translational regulation protein is intrinsically disordered and is implicated in autism and neurodegenerative diseases. Proof-of-principle biophysical experiments such as Förster resonance energy transfer (FRET)and fluorescence correlation spectroscopy (FCS) were performed on the 4E-BP2 protein labelled using our new method. The results highlight the quality of the novel sample preparation technique, as well as help reveal new dynamic information about the disordered C-terminal region of this protein.

Constrained Glu‐Gly and Gln‐Gly dipeptide surrogates from γ‐substituted α‐amino‐δ‐lactam synthesis

Ramakotaiah Mulamreddy and William D. Lubell*

Département de Chimie, Université de Montréal, C.P. 6128 Succursale Centre-Ville, Montréal H3C 3J7 QC, Canada

Abstract:

In the context of studies of biologically active peptides towards the design of peptidomimetics, alpha-amino-delta-lactams (Adl) have been used as conformationally constrained analogs in structure-activity relationships and biological studies [1]. For example, enhanced selectivity and oral bioavailability were achieved by replacement of the D-Phe-Pro residue by (S)-Adl-Gly in thrombin inhibitors [2]. Ring substituted Adl analogs have been pursued to mimic both backbone and side-chain geometry in peptide structures such as b-turns [3]. Our presentation will describe the synthesis of gamma-substituted Adl analogs [4].

1. Gillespie, P.; Cicariello, J.; Olson, G. L. Conformational analysis of dipeptide mimetics. Peptide Sci. 1997, 43, 191-217.
2. Semple, J. E. et al. Design, synthesis, and evolution of a novel, selective, and an orally bioavailable class of thrombin inhibitors: P1-argininal derivatives incorporating P3-P4 lactam sulfonamide moieties J. Med. Chem. 1996, 39, 4531.
3. Zydowsky, T. M.; Dellaria Jr, J. F.; Nellans, H. N. Efficient and versatile synthesis of dipeptide isosteres containing. gamma- or delta-lactams. J. Org. Chem. 1988, 53, 5607-5616.
4. Mulamreddy, R.; Lubell W. D. Constrained Glu‐Gly and Gln‐Gly dipeptide surrogates from γ‐substituted α‐amino‐δ‐lactam synthesis Peptide Sci. 2020, 12, e24149.

Animal venoms contain hundreds of molecules, the majority of these molecules have pharmacological activity. The medicinal uses of scorpion and snake venoms are well documented in folk remedies and in traditional Western and Chinese medicine (Harvey et al., 1998, Koh et al., 2006). The first AMP from animal venoms was discovered in 1967. Melittin is the main active component of Apis mellifera bee venom with anti-Gram + and Gram- activity but also anti-fungal, anti-parasitic, anti-HIV and anti-cancer activity but is highly toxic to mammalian cells (Fennel et al., 1967). Since then, nearly 2900 AMPs have been identified to date, of which more than 2400 (83%) have antibacterial activity and more than 1300 are derived from animal venoms (Wang et al., 2004; 2009; 2016). We have identified, isolated, and characterised a peptide from hymenopteran venom with gram-acting antibacterial and antitumour activity. In a structure-activity study using several synthetic analogues, the activity and stability of this peptide were improved. Indeed, these analogs inhibit the growth of Pseudomonas (P.) aeruginosa with a MIC ranging from 0.78 to 3.125 micromolar. The novel L- and D- peptides : i) don’t exhibit any hemotoxicity up to 10 mM; ii) they have a strong therapeutic index not only for Gram- bacteria P.aeruginosa (Therapeutic Index of 3200) and E. coli (T .I of 1600), (iii) they showed no cytotoxicity against normal human cells (HUVEC, IMR 90 and BEAS cells) at high concentration; iiv) stability assay showed that only D- aa analogs were not affected by proteases and human serum, demonstrating that they can be used without inactivation by digestive intestinal digestive enzymes; v) the use of HeLa TLR4/IL-8 Renilla Luciferase cell line showed that they inhibit the binding of Gram negative lipopolysaccharide (LPS) to TLR4 and the related inflammation in a dose-dependent way with IC50 ranging from 7.2 μM to 16.8 μM; vi) they do not induce bacterial resistance after 30 days, no variation in MIC being observed unlike in the case of imipenem and Gemifloxacin against P. aeruginosa which increased after exposure to the antibiotic for 10 days. The preliminary results showed that the antibacterial effect of the peptides is related to their ability to rapidly (within few minutes) permeabilize the bacterial membranes (European patent2020 EP19218273.1).