Controlled micelle conjugation via charged peptide amphiphiles

Abstract We report the first demonstration of nonionic detergent micelle conjugation and phase separation using purpose‐synthesized, peptide amphiphiles, C₁₀‐(Asp)₅ and C₁₀‐(Lys)₅. Clustering is achieved in two different ways. Micelles containing the negatively charged peptide amphiphile C₁₀‐(Asp)₅ are conjugated (a) via a water‐soluble, penta‐Lys mediator or (b) to micelles containing the C₁₀‐(Lys)₅ peptide amphiphile. Both routes lead to phase separation in the form of oil‐rich globules visible in the light microscope. The hydrophobic nature of these regions leads to spontaneous partitioning of hydrophobic dyes into globules that were found to be stable for weeks to months. Extension of the conjugation mechanism to micelles containing a recently discovered, light‐driven proton pump King Sejong 1‐2 (KS1‐2) demonstrates that a membrane protein may be concentrated using peptide amphiphiles while preserving its native conformation as determined by characteristic UV absorption. The potential utility of these peptide amphiphiles for biophysical and biomedical applications is discussed.     

Biosynthesis of homoarginine (hArg) and asymmetric dimethylarginine (ADMA) from acutely and chronically administered free <Emphasis Type="SmallCaps">l</Emphasis>-arginine in humans

Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of nitric oxide (NO) synthesis, whereas l-arginine (Arg) and l-homoarginine (hArg) serve as substrates for NO synthesis. ADMA and other methylated arginines are generally believed to exclusively derive from guanidine (N ^G)-methylated arginine residues in proteins by protein arginine methyltransferases (PRMTs) that use S-adenosylmethionine (SAM) as the methyl donor. l-Lysine is known for decades as a precursor for hArg, but only recent studies indicate that arginine:glycine amidinotransferase (AGAT) is responsible for the synthesis of hArg. AGAT catalyzes the formation of guanidinoacetate (GAA) that is methylated to creatine by guanidinoacetate methyltransferase (GAMT) which also uses SAM. The aim of the present study was to learn more about the mechanisms of ADMA and hArg formation in humans. Especially, we hypothesized that ADMA is produced by N ^G-methylation of free Arg in addition to the known PRMTs-involving mechanism. In knockout mouse models of AGAT- and GAMT-deficiency, we investigated the contribution of these enzymes to hArg synthesis. Arg infusion (0.5 g/kg, 30 min) in children (n = 11) and ingestion of high-fat protein meals by overweight men (n = 10) were used to study acute effects on ADMA and hArg synthesis. Daily Arg ingestion (10 g) or placebo for 3 or 6 months by patients suffering from peripheral arterial occlusive disease (PAOD, n = 20) or coronary artery disease (CAD, n = 30) was used to study chronic effects of Arg on ADMA synthesis. Mass spectrometric methods were used to measure all biochemical parameters in plasma and urine samples. In mice, AGAT but not GAMT was found to contribute to plasma hArg, while ADMA synthesis was independent of AGAT and GAMT. Arg infusion acutely increased plasma Arg, hArg and ADMA concentrations, but decreased the plasma hArg/ADMA ratio. High-fat protein meals acutely increased plasma Arg, hArg, ADMA concentrations, as well as the plasma hArg/ADMA ratio. In the PAOD and CAD studies, plasma Arg concentration increased in the verum compared to the placebo groups. Plasma ADMA concentration increased only in the PAOD patients who received Arg. Our study suggests that in humans a minor fraction of free Arg is rapidly metabolized to ADMA and hArg. In mice, GAMT and N ^G-methyltransferases contribute to ADMA and hArg synthesis from Arg, whereas AGAT is involved in the synthesis of hArg but not of ADMA. The underlying biochemical mechanisms remain still elusive.     

Lysine and homoarginine are closely interrelated metabolites in the rat

L-Lysine (Lys) and L-arginine (Arg), but not L-homoarginine (hArg), are proteinogenic amino acids. In healthy humans, oral administration of hArg increased the plasma concentration of Lys, suggesting Lys as a metabolite of hArg. In humans and animals, hArg is biosynthesized from Arg and Lys by arginine:glycine amidinotransferase (AGAT). In vitro, recombinant human arginase and bovine liver arginase I hydrolyzed hArg to Lys, suggesting Lys as a metabolite of hArg. The aim of the present study was to investigate whether changes in blood concentrations of hArg and Lys in old rats fed for 4 months with varied controlled experimental diets could suggest interconversion of these amino acids. Blood samples (n?=?253) were taken before (T0) and after 2 months (T2) and 4 months (T4) of the experiment. Plasma concentrations of Lys and hArg were determined by gas chromatography–mass spectrometry. The plasma hArg concentration markedly correlated with the plasma Lys concentration at all timepoints (r?≥?0.7, P?<?0.0001). Further analysis demonstrated that hArg and Lys are closely and specifically associated independently of experimental time/rat age and diet, suggesting that hArg and Lys are mutual metabolites in old rats. Based on the plasma concentration changes, the median yield of hArg from Lys was determined to be 0.17% at T0 and each 0.27% at T2 and T4. With a circulating concentration of about 3 µM, hArg a major metabolite of Lys in healthy humans. hArg supplementation is currently investigated as a cardioprotective means to improve impaired hArg synthesis. Present knowledge suggests that Lys rather than hArg supplementation may be even more favorable.

     

Rational Optimizations of the Marine-Derived Peptide Sungsanpin as Novel Inhibitors of Cell Invasion

Cancer metastasis, including cell invasion, is a major cause of poor clinical outcomes and death in numerous cancer patients. In recent years, many efforts have been made to develop potent therapeutic molecules from naturally derived peptides. Sungsanpin is a naturally derived lasso peptide that inhibits A549 cell invasion. We aimed to evaluate the potential of sungsanpin derivatives as candidates for anti-invasion drugs. We synthesized an analog of sungsanpin (Sun A) using a solid-phase peptide synthesis strategy (SPPS) and further modified its structure to improve its anti-invasion activity. All peptides were tested for their proliferative inhibition and anti-invasion activities in the A549 cell lines. Octapeptide S3 and cyclooctapeptide S4 upregulated the expression of TIMP-1 and TIMP-2 mRNA effectively and thus improved the inhibitory effect on the invasion of A549 cells. The two peptides can inhibit the invasion of A549 cells by up to 60 %, suggesting that they have potential as lead molecules for the development of peptide inhibitors.     

Bradykinin in Solid-phase Peptide Synthesis

Bradykinin (Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg; BK) occupies a special place in the history of the development of solid-phase peptide synthesis (SPPS). It was the first biologically-active peptide to be synthesized by Merrifield using the new method. Since that time many hundreds of BK analogs have been synthesized by SPPS. Certain of these analogs show potential as drug candidates. Dedicated to the memory of Bruce Merrifield. John Stewart (left) and RB Merrifield (right) with the first automatic peptide synthesizer.      

Development of the efficient preparation method for thermoresponsive elastin-like peptides using liquid-phase synthesis combined with fragment condensation strategy

Elastin-like peptides (ELPs) are synthetic peptides that mimic the characteristic hydrophobic amino acid repeat sequences of elastin and exhibit temperature-dependent reversible self-assembly properties. ELPs are expected to be used as temperature-responsive biomolecular materials across diverse industrial and research fields, and there is a requirement for a straightforward method to mass-produce them. Previously, we demonstrated that phenylalanine-containing ELP analogs, namely, (FPGVG)_n, can undergo coacervation with short chains (n = 5). The Fmoc solid-phase peptide synthesis method is one strategy used to synthesize these short ELPs. However, owing to its low reaction efficiency, an efficient method for preparing ELPs is required. In this study, efficient preparation of ELPs was investigated using a liquid-phase synthesis method with a hydrophobic benzyl alcohol support (HBA-tag). Because HBA-tags are highly hydrophobic, they can be easily precipitated by the addition of poor solvents and recovered by filtration. This property allows the method to combine the advantages of the simplicity of solid-phase methods and the high reaction efficiency of liquid-phase methods. By utilizing liquid-phase fragment condensation with HBA-tags, short ELPs were successfully obtained in high yield and purity. Finally, the temperature-dependent response of the ELPs generated through fragment condensation was assessed using turbidity measurements, which revealed a reversible phase transition. Consequently, the ELPs exhibited a reversible phase transition, indicating successful synthesis of ELPs via fragment preparation with tags. These findings provide evidence of the potential for mass production of ELPs using this approach.     

Design of a Tumor Homing Cell-Penetrating Peptide for Drug Delivery

The major drawbacks with conventional cancer chemotherapy are the lack of satisfactory specificity towards tumor cells and poor antitumor activity. In order to improve these characteristics, chemotherapeutic drugs can be conjugated to targeting moieties e.g. to peptides with the ability to recognize cancer cells. We have previously reported that combining a tumor homing peptide with a cell-penetrating peptide yields a chimeric peptide with tumor cell specificity that can carry cargo molecules inside the cells. In the present study, we have used a linear breast tumor homing peptide, CREKA, in conjunction with a cell-penetrating peptide, pVEC. This new chimeric peptide, CREKA–pVEC, is more convenient to synthesize and moreover it is better in translocating cargo molecules inside cancer cells as compared to previously published PEGA–pVEC peptide. This study demonstrates that CREKA–pVEC is a suitable vehicle for targeted intracellular delivery of a DNA alkylating agent, chlorambucil, as the chlorambucil–peptide conjugate was substantially better at killing cancer cells in vitro than the anticancer drug alone.     

Anchor-linked intermediates in peptide amide synthesis are caused by dimeric anchors on the solid supports

Cleavage and kinetic studies have been carried out using commercially obtained H-Tyr(tBu)-5-(4′-aminomethyl-3′,5′-dimethoxyphenoxy)valeric acid-TentaGelS (H-Tyr(tBu)-4-ADPV-TentaGelS) and H-Tyr (tBu)-4-ADPV-Ala-aminomethyl-resin (H-Tyr(tBu)-4-ADPV-AM-resin) prepared from commercially available resin and loaded with commercially available Fmoc-4-ADPV-OH amide anchor. Cleavage with pure trifluoroacetic acid (TFA) gave the intermediate H-Tyr-4-ADPV-NH₂, which was then degraded to H-Tyr-NH₂, and cleavage with TFA/dichloromethane (1:9) yielded H-Tyr-4-ADPV-NH₂ which could be isolated in preparative amounts. Cleavage reactions with ¹⁵N-labelled H-Ala-4-ADPV-[¹⁵N]-Gly-AM-resin yielded the intermediate H-Ala-4-ADPV-NH₂, which contained no ¹⁵N as demonstrated by ¹H-NMR. The analysis of the commercial Fmoc-4-ADPV-OH amide anchor showed the presence of Fmoc-4-ADPV-4-ADPV-OH as an impurity in high amounts. This dimeric anchor molecule is the cause of formation of the anchor-linked peptide intermediate obtained during the cleavage from the resin. The particularly high acid-lability of the amide bond between the two ADPV moieties was utilized to synthesize sidechain and C-terminally 4-ADPV protected pentagastrin on a double-anchor resin, and to cleave it using 5% trifluoroacetic acid in dichloromethane. This method may offer a new way for the synthesis of protected peptide amides with improved solubility to be used in fragment condensation.     

Automated solid-phase synthesis of metabolically stabilized triazolo-peptidomimetics

The use of 1,4-disubstituted 1,2,3-triazoles as trans-amide bond surrogates has become an important tool for the synthesis of metabolically stabilized peptidomimetics. These heterocyclic bioisosters are generally incorporated into the peptide backbone by applying a diazo-transfer reaction followed by CuAAC (click chemistry) with an α-amino alkyne. Even though the manual synthesis of backbone-modified triazolo-peptidomimetics has been reported by us and others, no procedure has yet been described for an automated synthesis using peptide synthesizers. In order to efficiently adapt these reactions to an automated setup, different conditions were explored, putting special emphasis on the required long-term stability of both the diazo-transfer reagent and the Cu(I) catalyst in solution. ISA·HCl is the reagent of choice to accomplish the diazo-transfer reaction; however, it was found instable in DMF, the most commonly used solvent for SPPS. Thus, an aqueous solution of ISA·HCl was used to prevent its degradation over time, and the composition in the final diazo-transfer reaction was adjusted to preserve suitable swelling conditions of the resins applied. The CuAAC reaction was performed without difficulties using [Cu (CH₃CN)₄]PF₆ as a catalyst and TBTA as a stabilizer to prevent oxidation to Cu(II). The optimized automated two-step procedure was applied to the synthesis of structurally diverse triazolo-peptidomimetics to demonstrate the versatility of the developed methodology. Under the optimized conditions, five triazolo-peptidomimetics (8–5 amino acid residues) were synthesized efficiently using two different resins. Analysis of the crude products by HPLC-MS revealed moderate to good purities of the desired triazolo-peptidomimetics (70–85%). The synthesis time ranged between 9 and 12.5 h.     

Determination of equilibria constants of arginine:glycine amidinotransferase (AGAT)-catalyzed reactions using concentrations of circulating amino acids

Arginine:glycine amidinotransferase (AGAT) catalyzes mainly two reactions that generate 1) L-homoarginine (hArg) from L-arginine and L-lysine (K_harg) and 2) guanidinoacetate (GAA) and L-ornithine from L-arginine and glycine (K_gaa). Previously, we found that pharmacological treatment of Becker muscular dystrophy (BMD) patients with metformin or L-citrulline resulted in antidromic effects on serum hArg and GAA concentrations, seemingly acting as an inhibitor and effector of AGAT activity, respectively. Here, we used data of this study as a model to determine K_harg and K_gaa values by using the concentrations of the participating amino acids measured in serum samples of the BMD patients. The study aimed to prove the general utility of this approach to investigate effects of amino acids and drugs on AGAT-catalyzed reactions in vivo in humans.

     

Reactive Impurities in Excipients: Profiling,Identification and Mitigation of Drug–Excipient Incompatibility

Reactive impurities in pharmaceutical excipients could cause drug product instability, leading to decreased product performance, loss in potency, and/or formation of potentially toxic degradants. The levels of reactive impurities in excipients may vary between lots and vendors. Screening of excipients for these impurities and a thorough understanding of their potential interaction with drug candidates during early formulation development ensure robust drug product development. In this review paper, excipient impurities are categorized into six major classes, including reducing sugars, aldehydes, peroxides, metals, nitrate/nitrite, and organic acids. The sources of generation, the analytical method for detection, the stability of impurities upon storage and processing, and the potential reactions with drug candidates of these impurities are reviewed. Specific examples of drug–excipient impurity interaction from internal research and literature are provided. Mitigation strategies and corrective measures are also discussed.     

Peptide synthesis catalysed by a haloalkaliphilic serine protease from the archaeon Natrialba magadii (Nep)

Aims: Haloarchaeal proteases function optimally in high salt (low water activity); thus, they offer an advantage over the nonhalophilic counterparts as biocatalysts for protease‐catalysed peptide synthesis. The haloalkaliphilic archaeon Natrialba magadii secretes a solvent‐tolerant protease, Nep (Natrialba magadii extracellular protease). In this work, the ability of Nep to catalyse peptide synthesis was examined. Methods and Results: The tripeptide Ac‐Phe‐Gly‐Phe‐NH₂ was synthesized using Ac‐Phe‐OEt and Gly‐Phe‐NH₂ substrates as building blocks in the presence of Nep, 30% (v/v) dimethyl sulfoxide (DMSO) and 1·5 or 0·5 mol l^?1 NaCl. Purification and identification of the peptide product was achieved by RP‐HPLC and ESI‐MS, respectively. The native as well as the recombinant enzyme produced in Haloferax volcanii (HvNep) was similarly effective as catalysts for the synthesis of this model tripeptide with yields of up to 60% and without secondary hydrolysis of the product. HvNep catalysed the synthesis of various tripeptides with preference for those having aromatic amino acids in the P1 site. Conclusion: Nep is able to catalyse peptide synthesis under different salt concentrations in the presence of DMSO. Significance and Impact of Study: The catalytic property of Nep in peptide synthesis combined with overproduction of this protease in Hfx. volcanii anticipates the potential applicability of this haloarchaeal protease in biotechnology.     

Formulation matters! A spectroscopic and molecular dynamics investigation on the peptide CIGB552 as itself and in its therapeutical formulation

Synthetic therapeutic peptides (STP) are intensively studied as new-generation drugs, characterized by high purity, biocompatibility, selectivity and stereochemical control. However, most of the studies are focussed on the bioactivity of STP without considering how the formulation actually used for therapy administration could alter the physico-chemical properties of the active principle. The aggregation properties of a 20-mer STP (Ac-His-Ala-Arg-Ile-Lys-D-Pro-Thr-Phe-Arg-Arg-D-Leu-Lys-Trp-Lys-Tyr-Lys-Gly-Lys-Phe-Trp-NH₂), showing antitumor activity, were investigated by optical spectroscopy and atomic force microscopy imaging, as itself (CIGB552) and in its therapeutic formulation (CIGB552TF). It has found that the therapeutic formulation deeply affects the aggregation properties of the investigated peptide and the morphology of the aggregates formed on mica by deposition of CIGB552 and CIGB552TF millimolar solutions. Molecular dynamics simulations studied the first steps of CIGB552 aggregation under physiological ionic strength conditions (NaCl 150 mM), showing that peptide oligomers, from dimers to tetramers, are preferentially formed in this environment. Interestingly, cell viability assays performed on H-460 cell lines indicate a major antiproliferative activity of the peptide in its therapeutic formulation with respect to the peptide aqueous solution.     

Development of membrane-active peptide therapeutics in oncology

Membrane-active peptides play an essential role in many living organisms and their immune systems and counter many infectious diseases. Many have dual or multiple mechanisms and can synergize with other molecules, like peptides, proteins, and small molecules. Although membrane-active peptides have been intensively studied in the past decades and more than 3500 sequences have been identified, only a few received approvals from the US Food and Drug Administration. In this review, we investigated all the peptide therapeutics that have entered the market or were subjected to preclinical and clinical studies to understand how they succeeded. With technological advancement (e.g., chemical modifications and pharmaceutical formulations) and a better understanding of the mechanism of action and the potential targets, we found at least five membrane-active peptide drugs that have entered preclinical/clinical phases and show promising results for cancer treatment. We summarized our findings in this review and provided insights into membrane-active anticancer peptide therapeutics.     

以短肽组装体为模板仿生矿化二氧化硅纳米管

目的 二氧化硅纳米管由于具有生物相容性好、光学性质优异等特殊性能在不同领域展现出良好的应用前景，其尺寸和形貌可显著影响材料性质。为制备尺寸较大的纳米管并拓展其在不同领域的应用，本研究选择尺寸较大的多肽组装体为模板，进行二氧化硅的仿生矿化。方法 以Bola型多肽Ac-KI₃VK-CONH₂自组装形成的尺寸较大（直径约40 nm）的纳米管为模板，通过仿生矿化的方法制备二氧化硅纳米材料。首先考察了多肽纳米管的稳定性，发现在稀释、加入有机溶剂和改变溶液pH值时，纳米管的形貌均被破坏，展示出较差的稳定性。在此基础上，以该多肽纳米管为模板，选择反应速率较快的正硅酸甲酯（TMOS）为前驱体仿生矿化二氧化硅纳米材料，探索了不同矿化条件对二氧化硅纳米管尺寸和形貌的影响。结果 以反应速率较快的TMOS为硅源、体积百分比浓度为1.11%~3.33%、溶液为中性或者弱碱性时能够矿化形成形貌和尺寸较大且分布均匀的二氧化硅纳米管。结论 通过选择合适的多肽组装体模板和反应速率快的TMOS为硅源，成功制备出了尺寸较大的二氧化硅纳米管，这为拓展其在不同领域的应用奠定了基础，具有重要意义。     

Comprehensive analysis of the molecular docking of small molecule inhibitors to the Aβ1–40 peptide and its Osaka-mutant: insights into the molecular mechanisms of Aβ-peptide inhibition

Abstract

Alzheimer’s disease (AD) is the most common form of age-related neurodegeneration occurs because of deposition of proteins in the form of extracellular plaques containing aggregated amyloid beta (Aβ) peptide and intracellular neurofibrillary tangles composed of aggregated microtubule-binding protein tau. Amyloid aggregation process can be enhanced by several familial AD-associated mutations in Aβ peptide. In this study, we have unravelled the interactions of 40 small molecule inhibitors with the Osaka-mutant of Aβ_1–40 peptide at atomic level and characterized modes of their binding to mutant Aβ by docking approaches. We have also compared docking energies of these inhibitors with Osaka-mutant with those previously determined for the wild-type and Iowa-mutant peptides and discussed in light of the peptide conformations and non-covalent interactions. We have also discussed inhibition mechanisms of these three peptides. Our analyses revealed that these small molecules can efficiently inhibit Osaka-mutant. The binding modes of drugs with these three peptides are markedly different and so are the mechanisms of inhibition of these three peptides. Overall analysis of the data reveals that binding energy of Iowa-mutant drug complex is lowest and most stable which is followed wild-type peptide-drug complex followed by Osaka-mutant drug complex.

Communicated by Ramaswamy H. Sarma     

Identification of FAK substrate peptides via colorimetric screening of a one‐bead one‐peptide combinatorial library

Focal adhesion kinase (FAK) is a protein tyrosine kinase that is associated with regulating cellular functions such as cell adhesion and migration and has emerged as an important target for cancer research. Short peptide substrates that are selectively and efficiently phosphorylated by FAK have not been previously identified and tested. Here we report the synthesis and screening of a one‐bead one‐peptide combinatorial library to identify novel substrates for FAK. Using a solid‐phase colorimetric antibody tagging detection platform, the peptide beads phosphorylated by FAK were sequenced via Edman degradation and then validated through radioisotope kinetic studies with [γ‐³²P] ATP to derive Michaelis–Menton constants. The combination of results gathered from both colorimetric and radioisotope kinase assays led to the rational design of a second generation of FAK peptide substrates. Out of all the potential peptide substrates evaluated, the most active was GDYVEFKKK with a K_M = 92 μM and a V_max = 1920 nmol/min/mg. Peptide substrates discovered within this study may be useful diagnostic tools for future kinase investigations and may lead to novel therapeutic agents. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Biophysical characterization of layer‐by‐layer synthesis of aptamer‐drug microparticles for enhanced cell targeting

Targeted delivery of drug molecules to specific cells in mammalian systems demonstrates a great potential to enhance the efficacy of current pharmaceutical therapies. Conventional strategies for pharmaceutical delivery are often associated with poor therapeutic indices and high systemic cytotoxicity, and this result in poor disease suppression, low surviving rates, and potential contraindication of drug formulation. The emergence of aptamers has elicited new research interests into enhanced targeted drug delivery due to their unique characteristics as targeting elements. Aptamers can be engineered to bind to their cognate cellular targets with high affinity and specificity, and this is important to navigate active drug molecules and deliver sufficient dosage to targeted malignant cells. However, the targeting performance of aptamers can be impacted by several factors including endonuclease‐mediated degradation, rapid renal filtration, biochemical complexation, and cell membrane electrostatic repulsion. This has subsequently led to the development of smart aptamer‐immobilized biopolymer systems as delivery vehicles for controlled and sustained drug release to specific cells at effective therapeutic dosage and minimal systemic cytotoxicity. This article reports the synthesis and in vitro characterization of a novel multi‐layer co‐polymeric targeted drug delivery system based on drug‐loaded PLGA‐Aptamer‐PEI (DPAP) formulation with a stage‐wise delivery mechanism. A thrombin‐specific DNA aptamer was used to develop the DPAP system while Bovine Serum Albumin (BSA) was used as a biopharmaceutical drug in the synthesis process by ultrasonication. Biophysical characterization of the DPAP system showed a spherical shaped particulate formulation with a unimodal particle size distribution of average size ～0.685 µm and a zeta potential of +0.82 mV. The DPAP formulation showed a high encapsulation efficiency of 89.4 ± 3.6%, a loading capacity of 17.89 ± 0.72 mg BSA protein/100 mg PLGA polymeric particles, low cytotoxicity and a controlled drug release characteristics in 43 days. The results demonstrate a great promise in the development of DPAP formulation for enhanced in vivo cell targeting. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:249–261, 2018     

Structure-function studies of chemokine-derived carboxy-terminal antimicrobial peptides

Recent reports which show that several chemokines can act as direct microbicidal agents have drawn renewed attention to these chemotactic signalling proteins. Here we present a structure-function analysis of peptides derived from the human chemokines macrophage inflammatory protein-3α (MIP-3α/CCL20), interleukin-8 (IL-8), neutrophil activating protein-2 (NAP-2) and thrombocidin-1 (TC-1). These peptides encompass the C-terminal α-helices of these chemokines, which have been suggested to be important for the direct antimicrobial activities. Far-UV CD spectroscopy showed that the peptides are unstructured in aqueous solution and that a membrane mimetic solvent is required to induce a helical secondary structure. A co-solvent mixture was used to determine solution structures of the peptides by two-dimensional ¹H-NMR spectroscopy. The highly cationic peptide, MIP-3α_51-70, had the most pronounced antimicrobial activity and displayed an amphipathic structure. A shorter version of this peptide, MIP-3α_59-70, remained antimicrobial but its structure and mechanism of action were unlike that of the former peptide. The NAP-2 and TC-1 proteins differ in their sequences only by the deletion of two C-terminal residues in TC-1, but intact TC-1 is a very potent antimicrobial while NAP-2 is inactive. The corresponding C-terminal peptides, NAP-2_50-70 and TC-1_50-68, had very limited and no bactericidal activity, respectively. This suggests that other regions of TC-1 contribute to its bactericidal activity. Altogether, this work provides a rational structural basis for the biological activities of these peptides and proteins and highlights the importance of experimental characterization of peptide fragments as distinct entities because their activities and structural properties may differ substantially from their parent proteins.     

p‐Methoxyphenol: A potent and effective scavenger for solid‐phase peptide synthesis

During the final step of t‐Boc/Bzl, solid‐phase peptide synthesis (SPPS)‐protecting groups from amino acids (aa) side chains must be removed from the target peptides during cleavage from the solid support . These reaction steps involve hydrolysis with hydrogen fluoride (HF) in the presence of a nucleophile (scavenger), whose function is to trap the carbocations produced during S_N1‐type reactions. Five peptide sequences were synthesised for evaluating p‐methoxyphenol effectiveness as a potent scavenger. After the synthesis, the resin–peptide was then separated into two equal parts to be cleaved using two scavengers: conventional reactive p‐cresol (reported in the literature as an effective acyl ion eliminator) and p‐methoxyphenol (hypothesised as fulfilling the same functions as the routinely used scavenger). Detailed analysis of the electrostatic potential map (EPM) revealed similarities between these two nucleophiles, regarding net atomic charge, electron density distribution, and similar pKa values. Good scavenger efficacy was observed by chromatography and mass spectrometry results for the synthesised molecules, which revealed that p‐methoxyphenol can be used as a potent scavenger during SPPS by t‐Boc/Bzl strategy, as similar results were obtained using the conventional scavenger.