Fast Solution-Phase and Liquid-Phase Peptide Syntheses (SolPSS and LPPS) Mediated by Biomimetic Cyclic Propylphosphonic Anhydride (T3P®).

The growing applications of peptide-based therapeutics require the development of efficient protocols from the perspective of an industrial scale-up. T3P® (cyclic propylphosphonic anhydride) promotes amidation in the solution-phase through a biomimetic approach, similar to the activation of carboxylic moiety catalyzed by ATP-grasp enzymes in metabolic pathways. The T3P® induced coupling reaction was applied in this study to the solution-phase peptide synthesis (SolPPS). Peptide bond formation occurred in a few minutes with high efficiency and no epimerization, generating water-soluble by-products, both using N-Boc or N-Fmoc amino acids. The optimized protocol, which was successfully applied to the iterative synthesis of a pentapeptide, also allowed for a decrease in the solvent volume, thus improving process sustainability. The protocol was finally extended to the liquid-phase peptide synthesis (LPPS), where the isolation of the peptide was performed using precipitation, thus also showing the suitability of this coupling reagent to this emerging technique.

Carbodiimide-Mediated Beckmann Rearrangement of Oxyma-B as a Side Reaction in Peptide Synthesis.

The suppression of side reactions is one of the most important objectives in peptide synthesis, where highly reactive compounds are involved. Recently, the violuric acid derivative Oxyma-B was introduced into peptide synthesis protocols as a promising additive to efficiently control the optical purity of the amino acids prone to racemization. However, we discovered a side reaction involving the Beckmann rearrangement of Oxyma-B during the coupling reaction, which compromises the yield and purity of the target peptides. Here, we present the investigation of the mechanism of this rearrangement and the optimization of the coupling reaction conditions to control it. These results can be taken into account for the design of novel efficient oxime-based coupling reagents.

Application of Af4-Multidetection to Liraglutide in Its Formulation: Preserving and Representing Native Aggregation.

Aggregation is among the most critical parameters affecting the pharmacological and safety profile of peptide Active Pharmaceutical Ingredients (APIs). For this reason, it is of utmost importance to define the exact aggregation state of peptide drugs, particularly when the API is marketed as a ready-to-use solution. Consequently, appropriate non-destructive techniques able to replicate the peptide environment must be employed. In our work, we exploited Asymmetrical Flow Field-Flow Fractionation (AF4), connected to UV, dRI, fluorescence, and MALS detectors, to fully characterize the aggregation state of Liraglutide, a peptide API used for the treatment of diabetes type 2 and chronic obesity. In previous studies, Liraglutide was hypothesized to assemble into hexa-octamers in phosphate buffer, but no information on its behavior in the formulation medium was provided up to now. The method used allowed researchers to work using formulation as the mobile phase with excellent recoveries and LoQ/LoD, discerning between stable and degraded samples, and detecting, when present, aggregates up to 108 Da. The native state of Liraglutide was assessed and found to be an association into pentamers, with a non-spherical conformation. Combined to benchmark analyses, the sameness study was complete and descriptive, also giving insight on the aggregation process and covalent/non-covalent aggregate types.

Stable and Biocompatible Monodispersion of C60 in Water by Peptides.

The lack of solubility in water and the formation of aggregates hamper many opportunities for technological exploitation of C60. Here, different peptides were designed and synthesized with the aim of monomolecular dispersion of C60 in water. Phenylalanines were used as recognizing moieties, able to interact with C60 through π-π stacking, while a varying number of glycines were used as spacers, to connect the two terminal phenylalanines. The best performance in the dispersion of C60 was obtained with the FGGGF peptidic nanotweezer at a pH of 12. A full characterization of this adduct was carried out. The peptides disperse C60 in water with high efficiency, and the solutions are stable for months both in pure water and in physiological environments. NMR measurements demonstrated the ability of the peptides to interact with C60. AFM measurements showed that C60 is monodispersed. Electrospray ionization mass spectrometry determined a stoichiometry of C60@(FGGGF)4. Molecular dynamics simulations showed that the peptides assemble around the C60 cage, like a candy in its paper wrapper, creating a supramolecular host able to accept C60 in the cavity. The peptide-wrapped C60 is fully biocompatible and the C60 "dark toxicity" is eliminated. C60@(FGGGF)4 shows visible light-induced reactive oxygen species (ROS) generation at physiological saline concentrations and reduction of the HeLa cell viability in response to visible light irradiation.

Synthesis of α/ß dipeptides containing linear or cyclic α-dehydro-ß-amino acids as scaffolds for bioactive compounds.

The synthesis of α/ß dipeptides containing linear or cyclic α-dehydro-ß-amino acids has been performed starting from alkylidene acetacetamides, which were obtained from α-amino esters via Ir-catalyzed allylic amination. Differently hindered carbonates were synthesized via a protocol involving chemoselective Luche's reduction, acylation, and allylic amination. Depending on the nature of the selected α-amino acid, we observed strong influence on the product regiochemistry due to the carbonate size and the amino-acid side chain. In particular, complete regioselectivity was observed in the aminic allylation of carbonates deriving from amino acids possessing a methylene unit in ß-position. On the contrary, methyl carbonates deriving from ß-branched amino acid afforded different results depending on the hindrance of the carbonate. Moreover, spontaneous cyclization was observed for carbamate-containing intermediates, allowing to obtain peptidomimetic polyfunctionalized dihydropyrimidine-2,4-dione. Finally, by inverting the order of reduction/acylation steps on the starting alkylidene acetoacetamides, the formation of polyfunctionalized 1,3-oxazinane-2,4-dione was obtained demonstrating the wide applications of these substrates for the preparation of bioactive peptidomimetics.

Investigation of the GnRH antagonist degarelix isomerization in biological matrices.

One of the main objectives of peptide drug design is the improvement of peptide pharmacokinetics with maintaining biological activity, which can be achieved by the complex modifications of the primary structure of the peptides. However, these changes often lead to the formation of peculiar impurities in the peptide drugs and their metabolites, which require the development of advanced analytical methods to properly assess their content. Here, we investigated the degradation of the potent long-acting GnRH antagonist degarelix in various biologic media by the tailor-made HPLC method, which allows precise determination of 5-Aph(Hyd)-degarelix isomer, an impurity found in the degarelix active pharmaceutical ingredient (API) during its manufacturing. Unexpectedly, we discovered a rapid and irreversible conversion of degarelix API into the corresponding hydantoin isomer in serum, suggesting that this impurity can be also a potential drug metabolite in vivo. This finding underlines the importance of the development of more accurate and performing analytical techniques to correctly characterize the chemical composition of the manufactured drugs and their behavior under physiological conditions.

The dual nature of biomimetic melanin.

Melanin-inspired nanomaterials offer unique photophysical, electronic and radical scavenging properties that are widely explored for health and environmental preservation, or energy conversion and storage. The incorporation of functional melanin building blocks in more complex nanostructures or surfaces is typically achieved via a bottom-up approach starting from a molecular precursor, in most cases dopamine. Here we demonstrate that indeed, the oxidative polymerization of dopamine, for the synthesis of melanin-like polydopamine (PDA), leads to the simultaneous formation of more than one nanosized species with different compositions, morphologies and properties. In particular, a low-density polymeric structure and dense nanoparticles (NP) are simultaneously formed. The two populations could be separated and analyzed in real time using a chromatographic technique free of any stationary phase (flow field fractionation, FFF). The results following the synthesis of melanin-like PDA showed that the NP are formed only during the first 6 hours as a result of a supramolecular self-assembly-driven polymerization, while the formation of the polymer continues for about 36 hours. The two populations were also separated and characterized using TEM, UV-vis absorption spectroscopy, fluorescence and light scattering spectroscopy, DLS, FTIR, ζ-potential measurements, gel electrophoresis and pH titrations. Interestingly, very different properties between the two populations were observed: in particular the polymer contains a higher number of catechol units (8 mmol g-1 -OH) with respect to the NP (1 mmol g-1 -OH) and presents a much higher antioxidant activity. The attenuation of light by NP is more efficient than that by the polymer especially in the Vis-NIR region. Moreover, while the NP scatter light with an efficiency up to 27% they are not fluorescent, and the polymer does not scatter light but shows an excitation wavelength-dependent fluorescence typical of multi-fluorophoric uncoupled systems.

Reversible Supramolecular Noncovalent Self-Assembly Determines the Optical Properties and the Formation of Melanin-like Nanoparticles.

The role of noncovalent supramolecular self-assembly in the formation of melanin-like NP, as well as the nature of the electronic transition at the basis of their unique optical properties, is strongly debated. Here we demonstrate that, during the first stage of formation of synthetic melanin, polydopamine (PDA), a small fraction of the molecular precursor dopamine (DA) is oxidized to quinone (Q) and a simple supramolecular charge-transfer (CT) adduct is formed thanks to the electron donor and electron acceptor properties of DA and Q, respectively. This adduct, also detectable by HPLC-MS, presents the broad absorption band in the red-NIR region typical of melanin-like materials. Importantly, its disaggregation upon dilution can be easily detected since it leads to the disappearance of the CT band, indicating the reversibility of the process. Moreover, the stability constant K of the CT adduct could be obtained using a simple association model.

Fast MacMillan's Imidazolidinone-Catalyzed Enantioselective Synthesis of Polyfunctionalized 4-Isoxazoline Scaffolds.

The enantioselective 1,3-dipolar cycloaddition of nitrones and arylpropionaldehydes to generate highly functionalized scaffolds for application in drug discovery was herein investigated. The use of a second-generation MacMillan catalyst as hydrochloride salt consistently accelerated the reaction speed, allowing a decrease in the reaction time up to >100 times, still affording 4-isoxazolines with good to excellent enantiomeric ratios at room temperature. As a proof of concept, further functionalization of the isoxazoline core through Pd-catalyzed cross-coupling was performed, generating differently functionalized chemical architectures in high yield.

A New Approach to Supramolecular Structure Determination in Pharmaceutical Preparation of Self-Assembling Peptides: A Case Study of Lanreotide Autogel.

The supramolecular structure in peptides' prolonged-released gel formulations is the most critical parameter for the determination of the pharmaceutical profile of the drug. Here, we report our investigation on lanreotide Autogel as a case study. For the first time, we describe the use of the pulsed field gradient (PFG) diffusion-ordered spectroscopy (DOSY) magic-angle spinning NMR to characterize the supramolecular self-assembly and molecular mobility of different samples of lanreotide Autogel formulations prepared according to different formulation protocols. The diffusion coefficient was used to calculate the hydrodynamic radii of supramolecular assemblies and build relative molecular models. DOSY data were integrated with NMR imaging (MRI) measurements and atomic force microscopy (AFM) imaging.

Therapeutic Peptides Targeting PPI in Clinical Development: Overview, Mechanism of Action and Perspectives.

Targeting protein-protein interactions (PPIs) has been recently recognized as an emerging therapeutic approach for several diseases. Up today, more than half a million PPI dysregulations have been found to be involved in pathological events. The dynamic nature of these processes and the involvement of large protein surfaces discouraged anyway the scientific community in considering them promising therapeutic targets. More recently peptide drugs received renewed attention since drug discovery has offered a broad range of structural diverse sequences, moving from traditionally endogenous peptides to sequences possessing improved pharmaceutical profiles. About 70 peptides are currently on the marked but several others are in clinical development. In this review we want to report the update on these novel APIs, focusing our attention on the molecules in clinical development, representing the direct consequence of the drug discovery process of the last 10 years. The comprehensive collection will be classified in function of the structural characteristics (native, analogous, heterologous) and on the basis of the therapeutic targets. The mechanism of interference on PPI will also be reported to offer useful information for novel peptide design.

Palladium Catalyst Recycling for Heck-Cassar-Sonogashira Cross-Coupling Reactions in Green Solvent/Base Blend.

The identification of a green, versatile, user-friendly, and efficient methodology is necessary to facilitate the use of Heck-Cassar-Sonogashira (HCS) cross-coupling reaction in drug discovery and industrial production in the pharmaceutical segment. The Heck-Cassar and Sonogashira protocols, using N-hydroxyethylpyrrolidone (HEP)/water/N,N,N',N'-tetramethyl guanidine (TMG) as green solvent/base mixture and sulfonated phosphine ligands, allowed to recycle the catalyst, always guaranteeing high yields and fast conversion under mild conditions, with aryl iodides, bromides, and triflates. No catalyst leakage or metal contamination of the final product were observed during the HCS recycling. To our knowledge, a turnover number (TON) up to 2375, a turnover frequency (TOF) of 158 h-1 , and a process mass intensity (PMI) around 7 that decreased around 3 after solvent, base, and palladium recovery, represent one of the best results to date using a sustainable protocol. The Heck-Cassar protocol using sSPhos was successfully applied to the telescoped synthesis of Erlotinib (TON: 1380; TOF: 46 h-1 ).

Ampicillin sodium: Isolation, identification and synthesis of the last unknown impurity after 60 years of clinical use.

Ampicillin, discovered in 1958, was the first broad spectrum semisynthetic penicillin introduced into the market. Despite its wide use not all the impurities have been identified to date. Herein, the last unknown impurity present in commercially available medicines was isolated and identified. This impurity that accounts up to 0.8 in area % by HPLC (EP 10.0) in the Reference Listed Drugs (RLD) was characterized and identified to be the 16-keto penicillin G. The structure was confirmed by comparison with a chemically synthesized sample. The determination of the Relative Response Factor (RRF) of the impurity respect to the parent drug allowed to recalculate the real amount that is consistently below the reporting threshold.

Integrin-mediated adhesive properties of neutrophils are reduced by hyperbaric oxygen therapy in patients with chronic non-healing wound.

In recent years, several studies suggested that the ability of hyperbaric oxygen therapy (HBOT) to promote healing in patients with diabetic ulcers and chronic wounds is due to the reduction of inflammatory cytokines and to a significant decrease in neutrophils recruitment to the damaged area. α4 and ß2 integrins are receptors mediating the neutrophil adhesion to the endothelium and the comprehension of the effects of hyperbaric oxygenation on their expression and functions in neutrophils could be of great importance for the design of novel therapeutic protocols focused on anti-inflammatory agents. In this study, the α4 and ß2 integrins' expression and functions have been evaluated in human primary neutrophils obtained from patients with chronic non-healing wounds and undergoing a prolonged HBOT (150 kPa per 90 minutes). The effect of a peptidomimetic α4ß1 integrin antagonist has been also analyzed under these conditions. A statistically significant decrease (68%) in ß2 integrin expression on neutrophils was observed during the treatment with HBO and maintained one month after the last treatment, while α4 integrin levels remained unchanged. However, cell adhesion function of both neutrophilic integrins α4ß1 and ß2 was significantly reduced 70 and 67%, respectively), but α4ß1 integrin was still sensitive to antagonist inhibition in the presence of fibronectin, suggesting that a combined therapy between HBOT and integrin antagonists could have greater antinflammatory efficacy.

Side chain effect in the modulation of αvß3/α5ß1 integrin activity via clickable isoxazoline-RGD-mimetics: development of molecular delivery systems.

Construction of small molecule ligand (SML) based delivery systems has been performed starting from a polyfunctionalized isoxazoline scaffold, whose αvß3 and α5ß1 integrins' potency has been already established. The synthesis of this novel class of ligands was obtained by conjugation of linkers to the heterocyclic core via Huisgen-click reaction, with the aim to use them as "shuttles" for selective delivery of diagnostic agents to cancer cells, exploring the effects of the side chains in the interaction with the target. Compounds 17b and 24 showed excellent potency towards α5ß1 integrin acting as selective antagonist and agonist respectively. Further investigations confirmed their effects on target receptor through the analysis of fibronectin-induced ERK1/2 phosphorylation. In addition, confocal microscopy analysis allowed us to follow the fate of EGFP conjugated α5ß1 integrin and 17b FITC-conjugated (compound 31) inside the cells. Moreover, the stability in water solution at different values of pH and in bovine serum confirmed the possible exploitation of these peptidomimetic molecules for pharmaceutical application.

Simple amphiphilic α-hydrazido acids: Rational design, synthesis, and in vitro bioactivity profile of a novel class of potential antimicrobial compounds.

Pursuing the search for a new class of structurally simple mimics of antimicrobial peptides, we optimized a short, cheap and high-yielding synthesis of mono-charged amphiphilic α-hydrazido acid derivatives. The most active derivatives furnished MICs that are among the best values reported in literature for synthetic amphiphilic membranolytic compounds. They exhibited a broad-spectrum in vitro activity against a variety of Gram-positive and Gram-negative bacteria, including two multidrug-resistant strains. In spite of the minimal cationic charge, the best compounds demonstrated to be selective toward bacterial cell membranes over mammalian cell membranes. The relationship between either the antibacterial or the hemolytic activity and the overall lipophilicity furnished an easy way to individuate the best dimensional range for the hydrophobic portions. The importance of a non-disrupted amphiphilicity was also demonstrated. Considering the bioactivity profile and the ease of synthesis, these chemically and proteolitically stable hydrochlorides are suitable for development of a new class of wide-spectrum antibiotics.

Novel insights into the chemistry of an old medicine: A general degradative pathway for penicillins from a piperacillin/tazobactam stability study.

Piperacillin is a broad spectrum beta-lactam antibiotic used in combination with tazobactam for hospital-related bacterial infections. The reconstituted solutions must respect the sub-visible and visible particles specifications. It was claimed that the reformulation containing EDTA/sodium citrate was able to control the formation of an insoluble impurity responsible for the formation of particulate matter observed using Ringer Lactate as diluent. The nature of the impurities formed during the degradative process of piperacillin/tazobactam combination has been herein investigated, by exploring the effect of added excipients and pH variations. The exact structure of the isolated dimeric impurity, the penicilloic acid-piperacillin dimer, was determined through complete characterization, allowing to propose a novel degradative general pathway for beta-lactam antibiotics. The presence of EDTA resulted unnecessary to contain the formation of the insoluble impurity, since the use of sodium citrate alone allowed to avoid this drawback. Finally, the proposed mechanism was successfully applied to the design of a novel, easy and high purity procedure for the synthesis of the acetylated penicilloic acid, known related substance of piperacillin.

New isoxazolidinone and 3,4-dehydro-ß-proline derivatives as antibacterial agents and MAO-inhibitors: A complex balance between two activities.

Among the different classes of antibiotics, oxazolidinone derivatives represent important drugs, since their unique mechanism of action overcomes commonly diffused multidrug-resistant bacteria. Anyway, the structural similarity of these molecules to monoamino oxidase (MAO) inhibitors, like toloxatone and blefoxatone, induces in many cases loss of selectivity as a major concern. A small library of compounds based on isoxazolidinone and dehydro-ß-proline scaffold was designed with the aim to obtain antibacterial agents, evaluating at the same time the potential effects of structural features on MAO inhibitory behaviour. The structural modification introduced in the backbone, starting from Linezolid model, lead to a significant loss in antibiotic activity, while a promising inhibitory effect could be observed on monoamino oxidases. These interesting results are also in agreement with docking experiments suggesting a good binding pose of the synthesized compounds into the pocket of the oxidase enzymes, in particular of MAO-B.

Dehydro-ß-proline Containing α4ß1 Integrin Antagonists: Stereochemical Recognition in Ligand-Receptor Interplay.

A novel class of dehydro-ß-proline-containing peptidomimetics, designed to be effective as α4ß1 integrin ligands, has been developed on the basis of the fundamental requirements for the interactions of these transmembrane receptors with bioactive ligands. Dehydro-ß-proline ring has been synthesized through an original pathway, involving ring closing metathesis of a diallylamino derivative. The synthesized products showed to be effective and selective as α4ß1 integrin antagonists and displayed IC50 values in the nanomolar range in cell adhesion inhibition assays and in VCAM-1-induced phosphorylation of extracellular-signal-regulated kinases. Significant activity was observed also toward the homologous integrin α4ß7, while they did not display any activity toward selected members of ß1, ß2, and ß3 families. A strong dependence on the stereochemistry of the heterocyclic central core could be observed. The great importance of α4ß1 integrin in chronic inflammatory and autoimmune diseases suggests a possible exploitation of these ligands as lead compounds for therapeutic tools development.