Study of the effect of the peptide loading and solvent system in SPPS by HRMAS-NMR.

The SPPS methodology has continuously been investigated as a valuable model to monitor the solvation properties of polymeric materials. In this connection, the present work applied HRMAS-NMR spectroscopy to examine the dynamics of an aggregating peptide sequence attached to a resin core with varying peptide loading (up to 80%) and solvent system. Low and high substituted BHAR were used for assembling the VQAAIDYING sequence and some of its minor fragments. The HRMAS-NMR results were in agreement with the swelling of each resin, i.e. there was an improved resolution of resonance peaks in the better solvated conditions. Moreover, the peptide loading and the attached peptide sequence also affected the spectra. Strong peptide chain aggregation was observed mainly in highly peptide loaded resins when solvated in CDCl3. Conversely, due to the better swelling of these highly loaded resins in DMSO, improved NMR spectra were acquired in this polar aprotic solvent, thus enabling the detection of relevant sequence-dependent conformational alterations. The more prominent aggregation was displayed by the VQAAIDYING segment and not by any of its intermediary fragments and these findings were also corroborated by EPR studies of these peptide-resins labelled properly with an amino acid-type spin probe.     

Polymer-assisted solution-phase (PASP) library synthesis of alpha-ketoamides

A parallel solution-phase library synthesis of alpha-ketoamides is described. The two-step library synthesis is accomplished using polymer-assisted solution-phase (PASP) synthesis techniques. This high-yielding, multi-step sequence utilizes sequestering resins for the removal of reactants, reactant by-products, and tagged reagents. The first step of the library synthesis utilizes PASP resins to mediate the amide coupling of an alpha-hydroxy acid with an amine. The second step uses PASP resins for the periodinane oxidation of the alpha-hydroxy acid to an alpha-ketoamide leaving highly pure products after simple filtration and evaporation.     

Discovery and optimization of pseudopeptide leads towards peptidomimetic drugs

Summary Progress in the identification of primary leads is increasingly obtained by the production of molecular diversity via the synthesis of peptide and non-peptide libraries. In this review, statistical considerations are made about the feasibility and reliability of peptide libraries. It is shown that the number of beads per peptide engaged in synthesis controls the relative concentration of any two peptide types, while the total amount of resin determines the feasibility of the complete library in relation to peptide size. Molecular modelling is used to estimate the conformational diversity. A preliminary analysis of libraries by NMR, MS, MS/MS and capillary electrophoresis is advocated. Examples are given of optimized peptide leads in hirudin, neurokinin, bradykinin and angiopeptin series. Finally, the relative effect of hydrogen-bond potential and overall lipophilicity on oral absorption is evaluted on neurokinin-1 and endothelin-1 receptor antagonists.     

Preparation of protected peptide amides using the Fmoc chemical protocol. Comparison of resins for solid phase synthesis.

Different resins were examined for their potential use in the solid phase synthesis of protected peptide amides using the 9-fluorenylmethoxycarbonyl (Fmoc) chemical protocol. The model protected peptide amide BocTyr-Gly-Gly-Phe-Leu-Arg(Pmc)NH2 (1) was synthesized on both the acid-labile 4-(2',4'-dimethoxyphenyl-Fmoc-aminomethyl)phenoxy resin (Rink amide resin) (2) and on resins containing the base-labile linker 4-hydroxymethylbenzoic acid. Of the resins examined only the methylbenzhydrylamine resin containing the 4-hydroxymethylbenzoic acid linkage, which was cleaved by ammonolysis in isopropanol, gave the model peptide 1 in good overall yield (53% including functionalization). Thus the synthesis of protected peptide amides by solid phase synthesis using Fmoc-protected amino acids with t-butyl-type side chain protecting groups is feasible. The choice of peptide-resin linkage and its cleavage conditions, however, are critical to the success of such syntheses. The potential application of this synthetic strategy to the preparation of novel peptide amides is discussed.     

Development of a herbicide biosensor using a peptide receptor screened from a combinatorial library

The purpose of this study was to screen for peptides that bind herbicides with a chlorinated aniline chemical structure. A tetrapeptide library was constructed using a solid phase split synthesis approach. Peptide beads were suspended in a buffer containing fluorescent-labeled dichloroaniline (DCA) as the bait. Eighteen fluorescent peptide beads were selected which bound to the bait after two rounds of staining screenings. The beads were then stained and suspended in a solution containing an excess of DCA and five quenched peptide beads were subsequently selected that recognized the DCA moiety. The screened peptides had many sequence similarities. The binding affinity of the screened peptides to herbicides was analyzed using surface plasmon resonance (SPR). N′-(3,4-dichlorophenyl)-N,N-dimethylurea [3-(3,4-dichlorophenyl)-1,1-dimethylurea] solution was injected over the peptide immobilized SPR chip. The SPR signal was found to increase in proportion to the DCMU concentration, whereas no signal was obtained from the negative control, 2-(2-methyl-4-chlorophenoxy) propionic acid (MCPP). From these results it is suggested that the screened peptide selectively recognizes the chemical structure of DCA.     

Microcontrolled release of biologically active compounds in chick embryos: beads of 200-microns diameter for the local release of retinoids

A method of controlled release that allows the continuous local application of retinoids (vitamin A derivatives) in living tissues has been developed. Several biocompatible 200-microns-diameter polymeric beads have been tested as possible carriers. Each type of bead was loaded by soaking in an isotopically labeled retinoid solution, washed, and then transferred into tissue culture medium for quantitative release measurements. Positively-charged ion-exchange resins of the Dowex 1 type were found to be the most suitable for the controlled release of retinoic acid, a negatively charged compound. For the controlled release of uncharged retinoids such as retinyl acetate, uncharged acrylic ester polymer beads are preferred; these beads can also be used to release the negatively charged compounds retinoic acid and prostaglandin E1. In all cases, a prolonged release is obtained that persists for more than a day. During this interval, the release is diffusion-controlled, and the total amount of compound released is directly proportional to the amount of the compound that the bead is exposed to during the initial loading step. High-performance liquid chromatography has been used to analyze the nature of the released retinoid. When the positively charged beads are loaded with all-trans-retinoic acid, there is a time-dependent decrease in the proportion of the all-trans isomer released which is due to an increased release of two cis isomers. This isomerization reaction occurs at a considerably slower rate when the uncharged beads are used as carriers. To mimic the conditions under which the local release of retinoic acid causes striking pattern duplications in developing chick wings, beads loaded with isotopically labeled retinoids were manually implanted into a slit cut into wing buds of stage-20 chick embryos. The release rate obtained was comparable to that found in vitro, and a time-dependent accumulation of the released radioactive compound was measured that was confined to the tissue near the site of implantation. All of the beads tested were readily accommodated by the tissue and could be easily removed at any time to terminate the treatment. It is believed that the controlled release of chemicals from such tiny biocompatible implants has a wide potential range of applications in biology.     

Peptide chemistry toolbox – Transforming natural peptides into peptide therapeutics

The development of solid phase peptide synthesis has released tremendous opportunities for using synthetic peptides in medicinal applications. In the last decades, peptide therapeutics became an emerging market in pharmaceutical industry. The need for synthetic strategies in order to improve peptidic properties, such as longer half-life, higher bioavailability, increased potency and efficiency is accordingly rising. In this mini-review, we present a toolbox of modifications in peptide chemistry for overcoming the main drawbacks during the transition from natural peptides to peptide therapeutics. Modifications at the level of the peptide backbone, amino acid side chains and higher orders of structures are described. Furthermore, we are discussing the future of peptide therapeutics development and their impact on the pharmaceutical market.     

α- 促黑激素的固相合成研究

目的:探索α-促黑激素的合成工艺。方法:采用多肽固相合成法制备α-促黑激素。以Rink amide-MBHA树脂为载体、使用Fmoc保护策略、TBTU、HOBt、DIEA为缩合剂体系,最后用TFA、苯甲硫醚、水、苯酚、乙二硫醇混合液将多肽从树脂上切割下来。结果:合成后的目标多肽产率达64.9%,经过RP-HPLC纯化纯度可达98%,质谱鉴定显示纯化产物与目标多肽理论相对分子质量一致。结论:该方法操作方便,反应结果稳定,为固相合成生产α-促黑激素提供了一种可行的工艺方案。     

Production of recombinant isotopically labelled peptide by fusion to an insoluble partner protein: generation of integrin αvβ6 binding peptides for NMR

The integrin αvβ6 is up-regulated in several cancers and has clinical potential for both tumour imaging and therapy. Peptide ligands have been developed which show good binding specificity for αvβ6 and provide an opportunity to study the interaction in more detail by NMR. Such studies ideally require (15)N and (13)C labelled peptides, and recombinant expression within E. coli provides a cost effective way of generating isotopically labelled proteins and peptides. In this study we have used an insoluble fusion partner (ketosteroid isomerase) to produce high yields of recombinant peptide. The insoluble nature of the fusion allowed simple product recovery by cell lysis and centrifugation, and thorough washing of the insoluble pellet to remove contaminating proteins avoided the need for nickel-affinity chromatography in denaturing conditions which is the standard procedure. The protocol described here is convenient to scale-up and requires only one chromatography step (reverse-phase HPLC) which is comparable to solid-phase synthesis.     

Catalytic activity of α-chymotrypsin in enzymatic peptide synthesis in ionic liquids

The catalytic activity of α-chymotrypsin in the enzymatic peptide synthesis of N-acetyl-l-tryptophan ethyl ester with glycyl glycinamide was examined in ionic liquids and organic solvents. The water content in 1-ethyl-3-methylimidazolium bis(fluorosulfonyl)imide ([emim][FSI]) affected the initial rates of peptide synthesis and hydrolysis. The activity of α-chymotrypsin was influenced by a kind of anions consisting of the same cation, [emim], when an ionic liquid was used as a solvent. The initial rate of peptide synthesis was improved 16-fold by changing from an organic solvent, acetonitrile, to an ionic liquid, [emim][FSI], at 25 °C. The activity of α-chymotrypsin in the peptide synthesis in [emim][FSI] was 17 times greater than that in acetonitrile at 60 °C, although the activity of α-chymotrypsin in the peptide synthesis gradually decreased with an increase in reaction temperature in [emim][FSI], similar to organic solvents. Moreover, α-chymotrypsin exhibited activity in [emim][FSI] and [emim][PF₆] at 80 °C.     

To Rink or Not to Rink Amide Link, that is the Question to Address for More Economical and Environmentally Sound Solid-Phase Peptide Synthesis

A comparative study is presented on the solid-phase peptide synthesis (SPPS) of the acyl carrier protein (ACP 65–74) sequence on a series of Rink amide resins possessing different matrix structures: poly(vinyl alcohol)-graft-poly(ethylene glycol) (PVA-g-PEG, 4), Tentagel-S-RAM (TG, 5), NovaGel (NG, 6), ChemMatrix (CM, 7) and polystyrene-divinylbenzene (PS-DVB, 8). In this comparison, the PEG-containing resins proved significantly better suited for the synthesis of pure ACP target sequence than the conventional PS-DVB solid supports (75–90% versus 52% crude purity). Amongst themselves, the PEG resins 4-7 exhibited similar capacity for providing pure peptide. Selecting PVA-g-PEG resin for a comparison of Rink amide linker versus no linker, the ACP (65–74) sequence was synthesized directly on the PVA-g-PEG resin 1, under identical conditions as employed in the synthesis on resin 4 bearing the Fmoc Rink linker, except for the final cleavage step, which was performed under more environmentally sound conditions using ester displacement with aqueous ammonia. Relative to its Rink amide counterpart 4, PVA-g-PEG resin 1 was cheaper to produce and possessed twice as much loading capacity (0.48 vs. 0.81 mmol/g). Moreover, Rink-less resin 1 gave higher yields of isolated pure peptide (61 vs. 45%) relative to its Fmoc Rink linker counterpart 4. In light of these results, the importance of the linker has been brought into question. As the need for large scale solid-phase peptide synthesis grows with greater demand for peptide products, ideal resins should be inexpensive to produce and employable under environmentally sound conditions to provide pure products. In this light, PVA-g-PEG resin 1 has demonstrated significant promise for economic and “green” SPPS.     

Use of commercial anion-exchange resins as solid support for peptide synthesis and affinity chromatography

This report demonstrates that due to the presence of residual reactive sites in their matrices, classical diethylaminoethyl-attaching commercial anion-exchanger resins such as DEAE-MacroPrep and DEAE-Sephadex A50 supports can be used for peptide synthesis. Moreover, due to the high stability of the peptide-resin bond in the final cleavage treatments, desired peptidyl-resins free of side-chain protecting groups, which enables them to be further used as solid support for affinity chromatography, can be obtained. To demonstrate this potentiality, a fragment corresponding to the antigenic and immunodominant epitope of sporozoites of the Plasmodium falciparum malaria parasite was synthesized in these traditional resins and antibody molecules generated against the peptide sequence were successfully retained in these peptidyl supports. Due to the maintenance of their original anion-exchange capacities, the present findings open the unique possibility of applying, simultaneously, dual anion-exchange and affinity procedures for purification of a variety of macromolecules.     

Role of integrins in regulation of collagen phagocytosis by human fibroblasts

Phagocytosis of collagen fibrils by fibroblasts is an important pathway for degradation of extracellular matrix in mature connective tissues. To study regulatory mechanisms in phagocytosis, 2-μm fluorescent beads coated with either collagen (COL) or bovine serum albumin (BSA) were incubated with human gingival fibroblasts in vitro. For these studies single cell suspensions were prepared by trypsinization, and bead internalization and collagen receptor expression were assessed by flow cytometry. After 3-h incubations, up to 8-fold more cells internalized COL beads than BSA-coated beads. Increased collagen coating concentration was associated with elevated proportions of cells that internalized COL beads, and was observed also in the presence of competing fibronectin-coated beads. The number of beads per cell and the percent of phagocytic cells increased proportionally with higher bead loadings. At > 4 beads per cell a maximum of ∼︁80% of cells were phagocytic. Cells reacted with mAbs against the α1, α2, and α3 integrin subunits were, respectively, 5%, 98% and 93% positively stained above background controls. All cells that internalized COL beads exhibited α2 staining but there were large proportions of phagocytic cells that were not stained for α1. In unfixed cells, bead internalization caused an immediate reduction of surface staining of membrane-bound α2 by ∼︁55% which returned to control levels within 3 h, indicating that cell-surface α2 was internalized by phagocytosis. Preincubation of cells with up to 8 COL beads per cell reduced the proportion of phagocytic cells and the number of internalized beads after a second COL bead incubation 4 h later. To assess the relationship between the percent of phagocytic cells and α2 integrin levels, serum starvation and cycloheximide experiments were conducted. Compared to controls, serum starvation for 24 h induced a 3.2-fold increase of cells internalizing COL beads but did not alter α2 staining levels. In contrast, 3 h cycloheximide treatment reduced α2 staining to 60% of control levels and this treatment also inhibited COL bead internalization. GRGDTP peptide as well as mAbs against the α1 and α2 subunits significantly reduced internalization of COL beads by 1.8 to 2.6-fold, whereas GRGESP peptide and α3 mAb exerted no effect. Internalization of BSA beads was not affected by any of these treatments. Collectively, these data indicate that the α2 integrin, along with other, as yet unidentified components, is likely involved in COL bead internalization. The α2 integrin subunit is rapidly recycled or synthesized following a phagocytic load. In contrast, the α1 integrin is not directly required for phagocytosis but may regulate the internalization step. © 1996 Wiley-Liss, Inc.     

QM/MM studies on the catalytic mechanism of Phenylethanolamine N-methyltransferase

Epinephrine is a naturally occurring adrenomedullary hormone that transduces environmental stressors into cardiovascular actions. As the only route in the catecholamine biosynthetic pathway, Phenylethanolamine N-methyltransferase (PNMT) catalyzes the synthesis of epinephrine. To elucidate the detailed mechanism of enzymatic catalysis of PNMT, combined quantum-mechanical/molecular-mechanical (QM/MM) calculations were performed. The calculation results reveal that this catalysis contains three elementary steps: the deprotonation of protonated norepinphrine, the methyl transferring step and deprotonation of the methylated norepinphrine. The methyl transferring step was proved to be the rate-determining step undergoing a SN2 mechanism with an energy barrier of 16.4 kcal/mol. During the whole catalysis, two glutamic acids Glu185 and Glu219 were proved to be loaded with different effects according to the calculations results of the mutants. These calculation results can be used to explain the experimental observations and make a good complementarity for the previous QM study.     

A high-yield method to extract peptides from rat brain tissue

A process to extract and enrich extracellular peptides and proteins from tissues should have broad utility in the burgeoning proteomics field. To address this need, a novel three-step protocol was developed to extract polypeptides from whole tissue samples and enrich the extracellular components. The initial homogenization of rat brain was carried out at neutral pH to optimize protein and peptide stability and solubility. Subsequent covalent chromatography on an activated thiopropyl resin was employed to debulk the tissue extract by selectively removing a substantial fraction of the intracellular protein component under nondenaturing conditions. Finally, extraction with 0.1% trifluoroacetic acid was used to selectively precipitate large proteins while enhancing the solubility of smaller proteins and peptides. The fractions from each step in the process were compared to a single extract obtained by homogenization in 0.5 M acetic acid. The recovery and yields of endogenous neuropeptides and an exogenously added peptide were evaluated by enzyme immunoassay and Western blotting, respectively. In summary, the three-step protocol was superior to the extraction of tissue with 0.5 M acetic acid in terms of peptide recovery, enrichment, and sample stability. Enrichment of the extracellular protein compartment from tissues should be valuable in proteomics experiments aimed at identifying biomarkers that can partition into serum.     

QM/MM studies on the catalytic mechanism of Phenylethanolamine N-methyltransferase

Epinephrine is a naturally occurring adrenomedullary hormone that transduces environmental stressors into cardiovascular actions. As the only route in the catecholamine biosynthetic pathway, Phenylethanolamine N-methyltransferase (PNMT) catalyzes the synthesis of epinephrine. To elucidate the detailed mechanism of enzymatic catalysis of PNMT, combined quantum-mechanical/molecular-mechanical (QM/MM) calculations were performed. The calculation results reveal that this catalysis contains three elementary steps: the deprotonation of protonated norepinphrine, the methyl transferring step and deprotonation of the methylated norepinphrine. The methyl transferring step was proved to be the rate-determining step undergoing a SN2 mechanism with an energy barrier of 16.4kcal/mol. During the whole catalysis, two glutamic acids Glu185 and Glu219 were proved to be loaded with different effects according to the calculations results of the mutants. These calculation results can be used to explain the experimental observations and make a good complementarity for the previous QM study.     

Solid-phase peptide synthesis: from standard procedures to the synthesis of difficult sequences

This protocol for solid-phase peptide synthesis (SPPS) is based on the widely used Fmoc/tBu strategy, activation of the carboxyl groups by aminium-derived coupling reagents and use of PEG-modified polystyrene resins. A standard protocol is described, which was successfully applied in our lab for the synthesis of the corticotropin-releasing factor (CRF), >400 CRF analogs and a countless number of other peptides. The 41-mer peptide CRF is obtained within approximately 80 working hours. To achieve the so-called difficult sequences, special techniques have to be applied in order to reduce aggregation of the growing peptide chain, which is the main cause of failure for peptide chemosynthesis. Exemplary application of depsipeptide and pseudoproline units is shown for synthesizing an extremely difficult sequence, the Asn(15) analog of the WW domain FBP28, which is impossible to obtain using the standard protocol.     

Isopeptide ligation catalyzed by quintessential sortase A: mechanistic cues from cyclic and branched oligomers of indolicidin

The housekeeping transpeptidase sortase A (SrtA) from Staphyloccocus aureus catalyzes the covalent anchoring of surface proteins to the cell wall by linking the threonyl carboxylate of the LPXTG recognition motif to the amino group of the pentaglycine cross-bridge of the peptidoglycan. SrtA-catalyzed ligation of an LPXTG containing polypeptide with an aminoglycine-terminated moiety occurs efficiently in vitro and has inspired the use of this enzyme as a synthetic tool in biological chemistry. Here we demonstrate the propensity of SrtA to catalyze "isopeptide" ligation. Using model peptide sequences, we show that SrtA can transfer LPXTG peptide substrates to the ε-amine of specific Lys residues and form cyclized and/or a gamut of branched oligomers. Our results provide insights about principles governing isopeptide ligation reactions catalyzed by SrtA and suggest that although cyclization is guided by distance relationship between Lys (ε-amine) and Thr (α-carboxyl) residues, facile branched oligomerization requires the presence of a stable and long-lived acyl-enzyme intermediate.     

An on-bead assay for the identification of non-natural peptides targeting the androgen receptor-cofactor interaction

An efficient and rapid on-bead screening method was established to identify non-natural peptides that target the Androgen Receptor-cofactor interaction. Binding of the Androgen Receptor ligand binding domain to peptide sequences displayed on beads in a One-Bead-One-Compound format could be screened using fluorescence microscopy. The method was applied to generate and screen both a focussed and a random peptide library. Resynthesis of the peptide hits allowed for the verification of the affinity of the selected peptides for the Androgen Receptor in a competitive fluorescence polarization assay. For both libraries strong Androgen Receptor binding peptides were found, both with non-natural and natural amino acids. The peptides identified with natural amino acids showed great similarity in terms of preferred amino acid sequence with peptides previously isolated from biological screens, thus validating the screening approach. The non-natural peptides featured important novel chemical transformations on the relevant hydrophobic amino acid positions interacting with the Androgen Receptor. This screening approach expands the molecular diversity of peptide inhibitors for nuclear receptors.     

Cell fingerprint patterns using designed α-helical peptides to screen for cell-specific toxicity

We conducted cell-based cytotoxicity screening of a 101-membered α-helical peptide library using cell fingerprints (CFPs). The CFP data suggested that there is a relationship between cytotoxicity and peptide characteristics, such as hydrophobicity, charge, and amino acid composition. In spite of the small size of the library used in this study, several peptides demonstrated cell-specific toxicity. The strategy of combining a designed peptide library with CFP thus shows real promise for peptide-based screening with cells.