PEGA Resin Research Articles

Fluorescence-Based On-Resin Detection of Three Model Proteases

A new approach to on-resin detection of three model proteases (trypsin, chymotrypsin, and thrombin) has been developed, while at the same time already described methodology for simultaneous detection of two enzymes (trypsin and chymotrypsin) has been additionally generalized. Appropriate immobilized substrates, comprising specifically cleavable peptide sequences capped with fluorescent dyes, have been synthesized on Rink Amide PEGA resin or Amino PEGA resin modified with backbone amide linker (BAL). Resulting solid support-bound probes were then dispersed into Tris-HCl buffer solution (pH = 8.0) and subjected to enzymatic cleavage. Liberated fluorophores have been tracked by fluorescence measuring. The competitive activities of studied proteases towards the thrombin probe have been efficiently limited and controlled by employing a Bowman-Birk inhibitor into a system.

Solid Phase Synthesis of Thioether‐linked Peptide Macrocycles via Palladium‐Catalyzed Intramolecular S‐Arylation and S‐Alkenylation

AbstractA new protocol for synthesis of thioether‐linked peptide macrocycles via Pd‐catalyzed intramolecular S‐arylation and S‐alkenylation on solid phase is developed. The use of PEGA resin in mixed solvents of CH3CN and H2O is critical to suppress the undesired S‐arylation reactions with the XantPhos‐Pd‐G3 catalyst. Peptide macrocycles of varied ring size, composition, and with different thioether linkers can be prepared in moderate to good yield with one column chromatography purification.

Immobilized Fluorescent Probes for Simultaneous Multiple Protease Detection

A new concept for simultaneous detection of two model proteases based on immobilized fluorescently labelled peptides was developed and evaluated. Each probe was composed of a carrier modified by poly(ethylene glycol) (PEG) chains, a specifically cleavable linker, and a fluorescent dye incorporated in the peptide tail. Based on a single excitation–double emission fluorescence response of liberated fluorophores caused by enzymatic cleavage, the presence of a single or both proteases in a mixture was unambiguously determined in an experimentally established concentration range. Among the tested solid supports, Rink Amide PEGA resin was recognized as the most suitable choice from the perspective of on-resin enzyme assays.

Attachment of cyclodextrin acids to PEGA resin and study of binding with fluorescence microscopy

Three different cyclodextrin acids, 6A,6D-di-O-(prop-2-carboxy-1,3-dienyl)-α-cyclodextrin (1), 6-deoxy-β-cyclodextrin-6-carboxylic acid (2), 6-deoxy-β-cyclodextrin-6-ethylenecarboxylic acid (3), were prepared and attached to amino PEGA resin as amides using coupling conditions with COMU and NEM. Host-guest binding to the resins was studied by fluorescence microscopy using 8-anilinoaphtalene-1-sulfonic acid (ANS) as guest, and was found to follow the equation IF = IFmax*[ANS]/([ANS] + Kd) where F, Fmax and Kd are the fluorescence, maximum fluorescence and Kd the dissociation constant for the ANS-cyclodextrin complex, respectively. Kd was 4.4, 2.4 and 4.9 × 10−4 M for the three resins. Competitive inhibition of ANS binding was performed with 1-adamantanylamine and octyl β-d-glucoside with the latter being selective for the α-cyclodextrin as expected.

Laccase aggregates via poly-lysine-supported immobilization onto PEGA resin, with efficient activity and high operational stability and can be used to degrade endocrine-disrupting chemicals

Laccase was for the first time immobilized as enzyme aggregates onto PEGA resin using the technique of poly-lysine-supported cross-linking. Immobilized laccase showed efficient enzymatic activity with high operational stability and good reusability.

Highly Functionalized β-Cyclodextrins by Solid-Supported Synthesis.

Using covalent capture, a high yielding selective mono-functionalization of heptakis-[6-deoxy-6-(2-aminoethylsulfanyl)]-β-CD with a 5-mercaptopentyl functional group has been achieved. Here, we demonstrate the immobilization of the mono-thiol functionalized β-CD on PEGA resin via a disulfide bond, enabling solid-phase elaboration of the remaining six primary amines. To showcase the potential of this method, the amines were elaborated to tripeptides through standard Fmoc-peptide chemistry. A small library of CD-tripeptide conjugates was generated which, when reduced from the solid support, could be tagged at the released thiol with an environmentally sensitive fluorophore. The resulting library of sensors showed potential for the differential sensing of various bile salts. The described methodology provides a rapid and versatile route to synthesize highly functionalized libraries of CD derivatives that may be tailored towards applications in sensing, catalysis, and multivalent displays.

Catch and release of concanavalin A by a mannose-immobilized photoaffinity PEGA resin coupled with a cleavable disulfide linker.

A photoaffinity PEGA resin containing mannose as a ligand and disulfide as a cleavable linker was prepared. The resin was crosslinked to concanavalin A, a binding protein of mannose, by UV irradiation, and the protein was subsequently released by cleavage of the disulfide linker.

Specificity studies on Kallikrein-related peptidase 7 (KLK7) and effects of osmolytes and glycosaminoglycans on its peptidase activity

KLK7 substrate specificity was evaluated by families of fluorescence resonance energy transfer (FRET) peptides derived from Abz-KLFSSK-Q-EDDnp (Abz=ortho-aminobenzoic acid and Q-EDDnp=glutaminyl-N-[2,4-dinitrophenyl] ethylenediamine), by one bead-one peptide FRET peptide library in PEGA resin, and by the FRET peptide libraries Abz-GXX-Z-XX-Q-EDDnp (Z and X are fixed and random natural amino acids, respectively). KLK7 hydrolyzed preferentially F, Y or M, and its S1′ and S2′ subsites showed selectivity for hydrophilic amino acids, particularly R and K. This set of specificities was confirmed by the efficient kininogenase activity of KLK7 on Abz–MISLM↓KRPPGFSPF↓RSSRI-NH2 (↓indicates cleavage), hydrolysis of somatostatin and substance P and inhibition by kallistatin. The peptide Abz-NLY↓RVE-Q-EDDnp is the best synthetic substrate so far described for KLK7 [kcat/Km=455 (mMs)−1] that was designed from the KLK7 substrate specificity analysis. It is noteworthy that the NLYRVE sequence is present in human semaphorin 6B. KLK7 is activated by GAGs, inhibited by neutral salts, and activated by high concentration of kosmotropic salt. Pyroglutamic acid inhibited KLK7 (Ki=33mM) and is present in skin moisturizing factor (124mM). The KLK7 specificity described here and elsewhere reflects its participation in patho-physiological events in skin, the gastrointestinal tract and central nervous system, where KLK7 is significantly expressed.

Solid‐Phase Synthesis and Biological Evaluation of N‐Dipeptido L‐Homoserine Lactones as Quorum Sensing Activators

Bacteria use small signaling molecules to communicate in a process termed "quorum sensing" (QS), which enables the coordination of survival strategies, such as production of virulence factors and biofilm formation. In Gram-negative bacteria, these signaling molecules are a series of N-acylated L-homoserine lactones. With the goal of identifying non-native compounds capable of modulating bacterial QS, a virtual library of N-dipeptido L-homoserine lactones was screened in silico with two different crystal structures of LasR. The 30 most promising hits were synthesized on HMBA-functionalized PEGA resin and released through an efficient acid-mediated cyclative release mechanism. Subsequent screening for modulation of QS in Pseudomonas aeruginosa and E. coli identified six moderately strong activators. A follow-up library designed from the preliminary derived structure-activity relationships was synthesized and evaluated for their ability to activate the QS system in this bacterium. This resulted in the identification of another six QS activators (two with low micromolar activity) thus illuminating structural features required for QS modulation.

A convenient procedure for the solid-phase synthesis of hydroxamic acids on PEGA resins

An efficient method for the solid-phase synthesis of hydroxamic acids is described. The method comprises the nucleophilic displacement of esters immobilized on PEGA resins with hydroxylamine/sodium hydroxide in isopropanol. The hydroxyaminolysis protocol is compatible with a broad range of PEGA-supported peptide and peptidomimetic esters. The methodology was found to be compatible with two new strategies for the synthesis of solid-supported lactams and diketopiperazines, respectively, both relying on the high inter- and intramolecular reactivity of cyclic N-acyliminium ions with electron-rich aromatics and heteroaromatics, ultimately affording hydroxamic acid derivatives in high purities.

Solid-phase synthesis of an apoptosis-inducing tetrapeptide mimicking the Smac protein

An approach for the solid-phase synthesis of apoptosis-inducing Smac peptidomimetics is presented. Using a Rink linker strategy, tetrapeptides mimicking the N-4-terminal residue of the Smac protein [(N-Me)AVPF sequence] were synthesized on PEGA resin in excellent purities and yields. Following two synthetic routes, a known tetrapeptide, incorporating a substituted proline, previously shown to exhibit excellent biological activity in vitro as well as low toxicity, was synthesized effectively on a solid support.

Metabolically Stable Cellular Adhesion to Inert Surfaces

The structure of D-amino acid hexapeptides that promote cellular adhesion was determined by screening D-amino acid hexapeptide libraries synthesized on otherwise inert beaded PEGA resin. These new adhesion molecules provide a completely stable cellular environment and facilitate the maintenance of a monolayer of cells on beads for extended periods. The presence of the peptides promotes spreading of the cells on the bead surface. Not surprisingly, the molecules contained a significant number of arginines and/or lysines. However, the exact structure of each peptide is quite important for the degree of adhesion observed, and a motif with three or four basic amino acids spaced within amino acids of intermediate polarity clearly prevailed, for example, k-l/r-h-r-i/v-r-a; this maintains a polar/hydrophobic balance.

Click to Join Peptides/Proteins Together

Copper(I) is able to catalyze Huisgen 1,3-dipolar cycloaddition in a "click" fashion. This copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction presents excellent chemoselectivity and occurs over a wide-range of reaction conditions. It shows tolerance to variation in both pH and solvent polarity, thereby facilitating the ligation of peptides and proteins to produce peptidomimetics and synthetic proteins. In addition, the only product formed is a 1,4-disubstituted-1,2,3-triazole moiety, in many aspects resembling the natural peptide bond, including hydrogen-bonding capability, planarity, distance between the 1 and 4 substituents, and conformational restriction of the peptide backbone; thus the triazole-backbone-modified peptide, in which a triazole replaces the amide bond, may be anticipated to present a secondary structure similar to that of its natural counterpart. This Focus Review describes the scope and applications of copper(I)-catalyzed alkyne–azide cycloaddition in synthetic peptide/protein chemistry.

Improved Fmoc‐based solid‐phase synthesis of homologous peptide fragments of human and mouse prion proteins

The synthesis of difficult peptide sequences has been a challenge since the very beginning of SPPS. The self-assembly of the growing peptide chains has been proposed as one of the causes of this synthetic problem. However, there is an increasing need to obtain peptides and proteins that are prone to aggregate. These peptides and proteins are generally associated with diseases known as amyloidoses. We present an efficient SPPS of two homologous peptide fragments of HuPrP (106-126) and MoPrP105-125 based on the use of the PEGA resin combined with proper coupling approaches. These peptide fragments were also studied by CD and TEM to determine their ability to aggregate. On the basis of these results, we support PEG-based resins as an efficient synthetic tool to prepare peptide sequences prone to aggregate on-resin.

Screening of polymeric supports and enzymes for the development of an endo enzyme cleavable linker

Several polymeric supports possessing an ester moiety were prepared and a range of enzymes was investigated to hydrolyse the ester linkage and release a signalling group into solution for applications in immunoassays. Pseudomonas lipases were found to most readily cleave the solution-phase analogue and this observation translated well to the corresponding polymeric supports, where the most effective were PEGA resins and the LPOS support PEG-6000.

Enzymatic synthesis of peptides on a solid support

We have previously shown that dipeptides can be synthesised in high yields from amino acids using protease catalysis in aqueous media, if the amino component is immobilised on porous PEGA resin (a copolymer of polyethylene glycol and polyacrylamide). Here we explore the scope of this methodology for using protected and glycosylated amino acids as well as the synthesis of longer peptides on resin and show that such a method can also be applied on non-porous surfaces, in particular on gold.

Identification of Small Molecule Binding Molecules by Affinity Purification Using a Specific Ligand Immobilized on PEGA Resin

We investigated the application of resins used in solid-phase synthesis for affinity purification. A synthetic ligand for FK506-binding protein 12 (SLF) was immobilized on various resins, and the binding assays between the SLF-immobilized resins and FK506-binding protein 12 (FKBP12) were performed. Of the resins tested in this study, PEGA resin was the most effective for isolating FKBP12. This matrix enabled the isolation of FKBP12 from a cell lysate, and the identification of SLF-binding peptides from a phage cDNA library. We confirmed the interaction between SLF and these peptides using a cuvette type quartz crystal microbalance (QCM) apparatus. Our study suggests that PEGA resin has great potential as a tool not only for the purification and identification of small-molecule binding proteins but also for the selection of peptides that recognize target molecules.

Solid-Phase Conversion of Aromatic Acetylenes to Carbonyl Compounds

Solid-phase conversion of aromatic acetylenes 1 to the corresponding carboxylic acids 2, α-ketocarboxylic acids 3, and methyl ketones 4 is described. Resin-bound TMS-acetylenes 1 were prepared from PEGA resin by Fmoc-based peptide synthesis. The desilylation of 1 with TBAF (4 equiv) followed by OsO4/NMO/HMTA-mediated oxidative cleavage to give the carboxylic acids 2. α-Ketocarboxylic acids 3 were obtained by direct treatment of 1 with OsO4/NMO/HMTA and ketones 4 by acid-mediated hydration of 1 using aqueous TFA.

Direct Site-Selective Covalent Protein Immobilization Catalyzed by a Phosphopantetheinyl Transferase

Immobilization of proteins onto solid supports is important in the preparation of functional protein microarrays and in the development of bead-based bioassays, biosensors, and industrial biocatalysts. In order to generate the stable, functional, and homogeneous materials required for these applications, attention has focused on methods that enable the efficient and site-specific covalent immobilization of recombinant proteins onto a wide range of platforms. To this end, the phosphopantetheinyl transferase Sfp was employed to catalyze the direct immobilization of recombinant proteins bearing the small, genetically encoded ybbR tag onto surfaces functionalized with CoA. Using mass spectrometry, it was shown that the Sfp catalyzes immobilization of a model acyl carrier protein (ACP) onto CoA-derivatized PEGA resin beads through specific covalent bond formation. Luciferase (Luc) and glutathione-S-transferase (GST) ybbR-fusion proteins were similarly immobilized onto PEGA resin retaining high levels of enzyme activity. This strategy was also successfully applied for the immobilization of the ACP, as well as ybbR-Luc, -GST, and -thioredoxin fusion proteins, on hydrogel microarray slides. Overall, the Sfp-catalyzed surface ligation is mild, quantitative, and rapid, occurring in a single step without prior chemical modification of the target protein. Immobilization of the target proteins directly from a cell lysate mixture was also demonstrated.

Optically Active (Peptido‐carbene)palladium Complexes: Towards True Solid‐Phase Combinatorial Libraries of Transition Metal Catalysts

AbstractThis work explores the potential of forming transition metal catalysts by combinatorial solid‐phase peptide chemistry to produce catalysts with enzyme‐like properties of stereoselectivity, regioselectivity and even substrate selectivity. A series of new functionalised carbene precursors/donors – imidazolium salts – each containing both amino acid and carboxylic acid functionality was synthesised in solution. The readily accessible carbene precursors were incorporated within the backbones of peptides attached to PEGA resin, by standard solid‐phase peptide coupling techniques. The synthetic strategy gave easy access to both mono‐ and didentate ligand systems, providing folded structures around the central transition metal atoms, with different degrees of steric congestion and bite angles. Changing the number of incorporated amino acids between the two carbene donors facilitated variation of the properties of the complexes. Both ligand systems were complexed to palladium(II) by standard base treatment, and the Pd complexes were studied by mass spectrometry and NMR spectroscopy. The monodentate (carbene)palladium complexes were each the product of enolisation of a neighbouring carbonyl group and loss of a proton, followed by coordination of the oxy anion to the palladium atom. The established methods are suitable for the combinatorial synthesis of palladium catalysts on solid support. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2008)