O‐Acyl isopeptide method: development of an O‐acyl isodipeptide unit for Boc SPPS and its application to the synthesis of Aβ1‐42 isopeptide

The O‐acyl isopeptide method was developed for the efficient preparation of difficult sequence‐containing peptide. Furthermore, development of the O‐acyl isodipeptide unit for Fmoc chemistry simplified its synthetic procedure by solid‐phase peptide synthesis. Here, we report a novel isodipeptide unit for Boc chemistry, and the unit was successfully applied to the synthesis of amyloid β peptide. Combination of Boc chemistry and the isodipeptide unit would be an effective method for the synthesis of many difficult peptides. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Combinatorial solid phase peptide synthesis and bioassays

Solid phase peptide synthesis method, which was introduced by Merrifield in 1963, has spawned the concept of combinatorial chemistry. In this review, we summarize the present technologies of solid phase peptide synthesis (SPPS) that are related to combinatorial chemistry. The conventional methods of peptide library synthesis on polymer support are parallel synthesis, split and mix synthesis and reagent mixture synthesis. Combining surface chemistry with the recent technology of microelectronic semiconductor fabrication system, the peptide microarray synthesis methods on a planar solid support are developed, which leads to spatially addressable peptide library. There are two kinds of peptide microarray synthesis methodologies: pre-synthesized peptide immobilization onto a glass or membrane substrate and in situ peptide synthesis by a photolithography or the SPOT method. This review also discusses the application of peptide libraries for high-throughput bioassays, for example, peptide ligand screening for antibody or cell signaling, enzyme substrate and inhibitor screening as well as other applications.     

Fullerene-based amino acids and peptides.

Recent advances in the chemistry of fullerene have allowed the synthesis of many classes of novel fullerene derivatives. Among these classes, fullerene-based amino acids and peptides are particularly interesting, both for structural studies and biological applications. In this review, we will discuss our own achievements in this rapidly growing field. In particular, the application of fulleroproline (Fpr) amino acids and peptides to medicinal chemistry and material science will be highlighted.     

Microreactors for peptide synthesis: looking through the eyes of twenty first century !!!

The twenty first century has witnessed several advances in synthetic chemistry, among them microreactors. It is expected that these devices will have a considerable impact on synthetic organic chemistry since they offer a wide range of applications in various fields. Perhaps the synthesis of peptides deserves mention in this regard as these molecules are emerging as therapeutics and offer several advantages over the so-called small molecules. This minireview does not aim to address microreactors in detail, but explains various peptide synthesis methods that involve microfluidic techniques, highlighting the need for further improvement and expansion of microdevices/microreactors.     

Advances in Fmoc solid‐phase peptide synthesis

Today, Fmoc SPPS is the method of choice for peptide synthesis. Very‐high‐quality Fmoc building blocks are available at low cost because of the economies of scale arising from current multiton production of therapeutic peptides by Fmoc SPPS. Many modified derivatives are commercially available as Fmoc building blocks, making synthetic access to a broad range of peptide derivatives straightforward. The number of synthetic peptides entering clinical trials has grown continuously over the last decade, and recent advances in the Fmoc SPPS technology are a response to the growing demand from medicinal chemistry and pharmacology. Improvements are being continually reported for peptide quality, synthesis time and novel synthetic targets. Topical peptide research has contributed to a continuous improvement and expansion of Fmoc SPPS applications. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Recent synthesis of aminophosphonic acids as potential biological importance

Aminophosphonic acids are an important group of medicinal compounds, and their synthesis has been a focus of considerable attention in synthetic organic chemistry as well as medicinal chemistry. Although the phosphonic and carboxylic acid groups differ considerably with respect to shape, size, and acidity, α-aminophosphonic acids are considered to be structural analogues of the corresponding amino acids and the transition state mimics peptide hydrolysis. This review summarizes recent developments in the synthesis, characterization and biological activity of α-aminophosphonic acid and N-analogues. An account of both uses will be presented, emphasizing one of the potential future developments, and some implications in medicinal chemistry are also disclosed. In addition, a brief account on the characterization of N-(phosphonomethyl) glycine derivatives will be presented.     

What to synthesize? From Emil Fischer to peptidomics.

The driving forces, incentives and strategic targets of peptide synthesis have undergone considerable evolution during the centenary following the pioneer work of Emil Fischer. In those days peptide synthesis was considered as a way of confirming the polypeptide theory of protein structure. The scientific community also expected (naively) that the synthesis would eventually lead to the creation of artificial living organisms. Only in the 1950s, when the first exact amino acid sequences were established did peptide chemistry obtain firmer ground and clearly defined targets. The total synthesis of peptide hormones and antibiotics became possible, providing valuable material for elucidating structure-functional relationships and the mechanisms of biological action. In the following years the number of peptides isolated from various biological sources grew with impressive speed and peptides became known as the most abundant, ubiquitous group of low molecular bioregulators. The design and synthesis of novel peptide based pharmaceuticals became an important area of peptide chemistry. At present we are facing the challenge of analysing the structures and bioactivities of total sets of peptides, i.e. peptidoms, present in concrete tissues or groups of cells. The results obtained along these lines at the IBCH RAS Institute of Bioorganic Chemistry are briefly considered in the review.     

Molecular engineering of antimicrobial peptides: microbial targets,peptide motifs and translation opportunities

The global public health threat of antimicrobial resistance has led the scientific community to highly engage into research on alternative strategies to the traditional small molecule therapeutics. Here, we review one of the most popular alternatives amongst basic and applied research scientists, synthetic antimicrobial peptides. The ease of peptide chemical synthesis combined with emerging engineering principles and potent broad-spectrum activity, including against multidrug-resistant strains, has motivated intense scientific focus on these compounds for the past decade. This global effort has resulted in significant advances in our understanding of peptide antimicrobial activity at the molecular scale. Recent evidence of molecular targets other than the microbial lipid membrane, and efforts towards consensus antimicrobial peptide motifs, have supported the rise of molecular engineering approaches and design tools, including machine learning. Beyond molecular concepts, supramolecular chemistry has been lately added to the debate; and helped unravel the impact of peptide self-assembly on activity, including on biofilms and secondary targets, while providing new directions in pharmaceutical formulation through taking advantage of peptide self-assembled nanostructures. We argue that these basic research advances constitute a solid basis for promising industry translation of rationally designed synthetic peptide antimicrobials, not only as novel drugs against multidrug-resistant strains but also as components of emerging antimicrobial biomaterials. This perspective is supported by recent developments of innovative peptide-based and peptide-carrier nanobiomaterials that we also review.     

Novel prodrug approach to photodynamic therapy: Fmoc solid-phase synthesis of a cell permeable peptide incorporating 5-aminolaevulinic acid

The first example of the synthesis of a peptide incorporating 5-aminolaevulinic acid (5-ALA) using standard Fmoc solid-phase chemistry is reported. The synthesised peptide contains residues 52-58 of the cell-permeable peptide Penetratin and represents a prototype for the enhanced topical delivery of 5-ALA using such oligopeptide vectors. Effective intracellular conversion of the peptide to the endogenous photosensitiser, protoporphyrin IX, is observed in PAM212 cells, thus demonstrating the potential of this approach for the development of novel peptide prodrugs for use in photodynamic therapy.     

O-N intramolecular acyl migration reaction in the development of prodrugs and the synthesis of difficult sequence-containing bioactive peptides

N-Ointramolecular acyl migration in Ser- or Thr-containing peptides is a well-known side reaction in peptide chemistry. It results in the mutual conversion of ester and amide bonds. Our medicinal chemistry study focused on the fact that the O-acyl product can be readily converted to the original N-acyl form under neutral or slightly basic conditions in an aqueous buffer and the liberated ionized amino group enhances the water solubility of O-acyl products. Because of this, we have developed a novel class of "O-N intramolecular acyl migration"-type water-soluble prodrugs of HIV-1 protease inhibitors. These prodrugs released the parent drugs via a simple chemical mechanism with no side reaction. In this study, we applied this strategy to important cancer chemotherapeutic agents, paclitaxel and its derivatives, to develop water-soluble taxoid prodrugs, and found that these prodrugs, 2'-O-isoform of taxoids, showed promising results with higher water solubility and proper kinetics in their parent drug formation by a simple pH-dependent chemical mechanism with O-N intramolecular acyl migration. These results suggest that this strategy would be useful in toxicology and medical economics. After the successful application of O-N intramolecular acyl migration in medicinal chemistry, this concept was recently used in peptide chemistry for the synthesis of "difficult sequence-containing peptides." The strategy was based on hydrophilic O-acyl isopeptide synthesis followed by the O-N intramolecular acyl migration reaction, leading to the desired peptide. In a model study with small, difficult sequence-containing peptides, synthesized "O-acyl isopeptides" not only improved the solubility in various media and efficiently performed the high performance liquid chromatography purification, but also altered the nature of the difficult sequence during SPPS, resulting in the efficient synthesis of O-acyl isopeptides with no complications. The subsequent O-N intramolecular acyl migration of purified O-acyl isopeptides afforded the desired peptides as precipitates with high yield and purity. Further study of the synthesis of a larger difficult sequence-containing peptide, Alzheimer's disease-related peptide (A beta 1-42), surprisingly showed that only one insertion of the O-acyl group drastically improved the unfavorable nature of the difficult sequence in A beta 1-42, and achieved efficient synthesis of 26-O-acyl isoA beta 1-42 and subsequent complete conversion to A beta 1-42 via the O-N intramolecular acyl migration reaction of 26-O-acyl isoA beta 1-42. This suggests that our new method based on O-N intramolecular acyl migration is an important method for the synthesis of difficult sequence-containing bioactive peptides.     

Lead identification to generate isoquinolinedione inhibitors of insulin-like growth factor receptor (IGF-1R) for potential use in cancer treatment

Insulin-like growth factor receptor (IGF-1R) is a growth factor receptor tyrosine kinase that acts as a critical mediator of cell proliferation and survival. This receptor is over-expressed or activated in tumor cells and is emerging as a novel target in cancer therapy. Efforts in our "Hit to Lead" group have generated a novel series of submicromolar IGF-1R inhibitors based on a isoquinolinedione template originating from a Lance enzyme HTS screen. Chemical triage and parallel synthesis incorporating focused library arrays were instrumental in moving these investigations through the Wyeth exploratory medicinal chemistry process. The strategies, synthesis, and SAR behind this interesting kinase scaffold will be described.     

Synthesis and characterization of two potential impurities (des-ethyl-Ganirelix) generated in the Ganirelix manufacturing process

Controlling certain diseases using peptide drugs has remarkably increased in the past two decades. In this regard, a generic formulation is an upfront solution to fulfill market demands. Ganirelix, a leading peptide active pharmaceutical ingredient (API) primarily used as a gonadotropin-releasing hormone antagonist (GnRH), has established a potential market value worldwide. But its generic formulation mandates detailed impurity profiles from a synthetic source and contemplates the sameness of a reference-listed drug (RLD). Post-chemical synthesis and processing of Ganirelix, some commercial sources have revealed two new potential impurities among many known, which show the deletion of an ethyl group from the hArg(Et)₂ residue at the sixth and eighth positions, named des-ethyl-Ganirelix. These impurities are unprecedented in traditional peptide chemistry, and such monoethylated-hArg building blocks are not easily accessible commercially to synthesize these two impurities. Here, we have outlined the synthesis, purification, and enantiomeric purity characterization of the amino acids and their incorporation in the Ganirelix peptide sequence to synthesize these potential peptide impurities. This methodology will enable the convenient synthesis of side-chain substituted Arg and hArg derivatives in peptide drug discovery platforms.     

Biosynthetic systems for nonribosomal peptide antibiotic assembly

Modular peptide synthetases, which act as the protein templates for the synthesis of a large number of peptide antibiotics and siderophores, hold great potential for the development of novel compounds. Recently, significant progress has been made towards understanding their molecular architecture and substrate specificity. The first crystal structure of a peptide synthetase has been solved, and the enzymes responsible for post-translational modification of peptide synthetases have recently been discovered. These will allow addressing important yet poorly understood mechanistic aspects.     

Poly-N-acrylylpyrrolidine. A new resin in peptide chemistry.

Entirely beaded poly-N-acrylylpyrrolidine-co-bisacrylyl-1,2-diaminoethane-co-N-acrylyl-1,6-diaminohexane.HCl(PAP), a new resin on which to perform peptide chemistry, has been prepared by reverse phase suspension polymerization in quantitative yield. In addition to being a superior support to polystyrene, albeit readily adaptable to current techniques of peptide synthesis, its versatility has been furthur extended by the introduction and use of new peptide-to-polymer linking groups, which allow the use of the bidirectional approach to peptide chemistry. One such linkage, which connects the side chain of cysteine to PAP via an acid resistant S-carbamoyl bond, was used in a bidirectional synthesis of deamino-oxytocin. PAP solvates and swells in solvents with wide-ranging polarities, including aqueous media. Thus, peptide coupling reactions were performed in organic media of high and of low polarity as well as in aqueous solution.     

Facile SPS of peptides having C-terminal Asn and Gln

Attachment of Fmoc-asparagine or glutamine to p-alkoxybenzyl alcohol type resins has always been difficult and not very effective. A very simple and effective method for the preparation of peptides terminating in asparagine or glutamine is described. The method involves quantitative attachment of Fmoc-Asp-OtBu or Fmoc-Glu-OtBu via their side-chain carboxyl group to a resin functionalized with our TMBPA linker for peptide amides. Peptide synthesis is performed using our standard Fmoc chemistry, and treatment with acid, e.g. TFA/DCM/scavenger mixtures, releases the Asn or Gln peptides.     

Design and synthesis of multifunctional gold nanoparticles bearing tumor-associated glycopeptide antigens as potential cancer vaccines

The development of vaccines against specific types of cancers will offer new modalities for therapeutic intervention. Here, we describe the synthesis of a novel vaccine construction prepared from spherical gold nanoparticles of 3-5 nm core diameters. The particles were coated with both the tumor-associated glycopeptides antigens containing the cell-surface mucin MUC4 with Thomsen Friedenreich (TF) antigen attached at different sites and a 28-residue peptide from the complement derived protein C3d to act as a B-cell activating "molecular adjuvant". The synthesis entailed solid-phase glycopeptide synthesis, design of appropriate linkers, and attachment chemistry of the various molecules to the particles. Attachment to the gold surface was mediated by a novel thiol-containing 33 atom linker which was further modified to be included as a third "spacer" component in the synthesis of several three-component vaccine platforms. Groups of mice were vaccinated either with one of the nanoplatform constructs or with control particles without antigen coating. Evaluation of sera from the immunized animals in enzyme immunoassays (EIA) against each glycopeptide antigen showed a small but statistically significant immune response with production of both IgM and IgG isotypes. Vaccines with one carbohydrate antigen (B, C, and E) gave more robust responses than the one with two contiguous disaccharides (D), and vaccine E with a TF antigen attached to threonine at the 10th position of the peptide was selected for IgG over IgM suggesting isotype switching. The data suggested that this platform may be a viable delivery system for tumor-associated glycopeptide antigens.     

Synthesis and construction of a novel multiple peptide conjugate system: strategy for a subunit vaccine design

We describe the design and synthesis of a novel well characterized multi-peptide conjugate (MPC) system containing antigens from human malaria parasite and the Tat protein of HIV type-1 (HIV-1-Tat). Construction of the MPC utilizes Fmoc solid-phase peptide synthesis coupled with solution chemistry. In the first phase, a core template that serves as primary anchor for the synthesis and attachment of multiple antigens is synthesized. Serine(trityl) and multiple lysine branches with epsilon groups blocked during chain assembly are incorporated forming a tetrameric core. Cysteine whose side chain thiol serves to couple haloacetyl or S-protected haloacetyl peptides is added to complete assembly of the core template. Modification to the coupling solvent, addition of key amino acid derivatives (N-[1-hydroxy-4-methoxybenzyl]) in the peptide sequence allows the synthesis of base peptides on the core template with molecular mass greater than 7500 kDa. Base peptides are then reacted with high performance liquid chromatography purified haloacetyl peptides to generate multiple peptide conjugates with molecular masses of 10 to 13 kDa. MPC constructs thus formed are further characterized by matrix assisted laser desorption-time of flight mass spectroscopy (MALDI-MS), amino acid analysis, size exclusion chromatography, and SDS-polyacrylamide gel electrophoresis (PAGE). To our knowledge, this is the first report describing a chemically well defined multiple conjugate system with potential for development of synthetic subunit vaccines.     

Click chemistry based rapid one-pot synthesis and evaluation for protease inhibition of new tetracyclic triazole fused benzodiazepine derivatives

We describe herein a one-pot synthesis of novel tetracyclic scaffolds that incorporate a fusion of a proline, 1,2,3-triazole ring with [1,4]-benzodiazepin-8(4H)-one ring systems following click chemistry. The expected peptide bond formation followed by in situ 1,3-dipolar cycloaddition in absence of any catalyst led to the formation of new triazole fused benzodiazepine derivatives.     

Optimized syntheses of Fmoc azido amino acids for the preparation of azidopeptides

The rise of CuI‐catalyzed click chemistry has initiated an increased demand for azido and alkyne derivatives of amino acid as precursors for the synthesis of clicked peptides. However, the use of azido and alkyne amino acids in peptide chemistry is complicated by their high cost. For this reason, we investigated the possibility of the in‐house preparation of a set of five Fmoc azido amino acids: β‐azido l ‐alanine and d ‐alanine, γ‐azido l ‐homoalanine, δ‐azido l ‐ornithine and ω‐azido l ‐lysine. We investigated several reaction pathways described in the literature, suggested several improvements and proposed several alternative routes for the synthesis of these compounds in high purity. Here, we demonstrate that multigram quantities of these Fmoc azido amino acids can be prepared within a week or two and at user‐friendly costs. We also incorporated these azido amino acids into several model tripeptides, and we observed the formation of a new elimination product of the azido moiety upon conditions of prolonged couplings with 2‐(1H‐benzotriazol‐1‐yl)‐1,1,3,3‐tetramethyluronium hexafluorophosphate/DIPEA. We hope that our detailed synthetic protocols will inspire some peptide chemists to prepare these Fmoc azido acids in their laboratories and will assist them in avoiding the too extensive costs of azidopeptide syntheses. Experimental procedures and/or analytical data for compounds 3 – 5 , 20 , 25 , 26 , 30 and 43 – 47 are provided in the supporting information. © 2017 The Authors Journal of Peptide Science published by European Peptide Society and John Wiley & Sons Ltd.     

The synthesis and spectroscopic analysis of the neurotoxic prion peptide 106-126: Comparative use of manual Boc and Fmoc chemistry

A peptide corresponding to residues 106-126 of the human prion protein (PrP) possesses the neurotoxic and amyloidogenic properties of the infectious form of the parental protein. This peptide is now identified as a 'difficult sequence' and synthesis using conventional manual Fmoc chemistry was unsuccessful with acylation terminating at a central core of hydrophobic amino acids. The use of tetramethylfluoroformamidinium hexafluorophosphate and 1-methyl-2- pyrrolidone as anti-aggregatory agents in the coupling steps improved the synthesis but still resulted in an incomplete peptide. The incorporation of N-(2-hydroxy-4-methoxybenzyl) protection at glycine residues 119 and 124 enabled synthesis of the full length peptide in low yield. Synthesis using Boc chemistry with in situ neutralisation gave the full length peptide in high yield.