Tools for investigating peptide-protein interactions: peptide incorporation of environment-sensitive fluorophores via on-resin derivatization

This protocol presents the peptide incorporation of environment-sensitive fluorophores derived from the dimethylaminophthalimide family. The procedure utilizes anhydride precursors of 4-dimethylaminophthalimide (4-DMAP) or 6-dimethylaminonaphthalimide (6-DMN), whose syntheses are described in a related protocol from these authors. In this protocol, the fluorophores are directly incorporated after solid-phase peptide synthesis (SPPS) via on-resin derivatization of peptides prepared using commercially available diamino acids, which are Alloc-protected on the side-chain amino group. The time required to complete the procedure depends on the size and number of peptides targeted. As an alternative to this approach, the corresponding fluorescent amino acids can be obtained in an Fmoc-protected form for convenient use as building blocks in SPPS. This option is described in a related protocol by these authors.     

Synthesis of phosphotyrosyl-containing phosphopeptides by solid-phase peptide synthesis.

The synthesis of phosphotyrosine-containing phosphopeptides using solid-phase peptide synthesis (SPPS) techniques is described. We present the synthesis of a Boc-phosphotyrosine derivative, which when used with modifications of the conventional SPPS protocol permits the incorporation of phosphotyrosine into synthetic peptides. The resulting phosphopeptides were authenticated by fast atom bombardment mass spectrometry, amino acid analysis, and phosphate assay. Alkaline phosphatase was found to dephosphorylate synthetic phosphopeptides at different rates, supporting the potential use of these synthetic substrates for studies of phosphoprotein phosphatases. Synthesis of a phosphopeptide using the described protocol has several advantages over the preparation of phosphopeptides via enzymatic phosphorylation.     

Development of a Method for the Solid-Phase Peptide Synthesis in Water

Various organic solvents are routinely used in peptide synthesis, safe disposal of which are now an important environmental problem. To circumvent this problem, during the last few years we focused on developing an organic solvent-free SPPS method using aqueous solvents. For the SPPS in water, we designed protected amino acids that could be used in the aqueous media. Here we described development of several types of water-soluble protected amino acids and their application to the SPPS in water, and a novel technology that uses water-dispersible protected amino acids for in-water peptide synthesis.     

Optimized selective N-methylation of peptides on solid support.

Peptides containing N(alpha)-methylamino acids exhibit interesting therapeutic profiles and are increasingly recognized as potentially useful therapeutics. Unfortunately, their synthesis is hampered by the high price and nonavailability of many N(alpha)-methylamino acids. An efficient and practical three-step procedure for selective N-methylation of peptides on solid support is described. The procedure was based on the well known solid-phase N-methylation of N(alpha)-arylsulfonyl peptides, which was improved by using dimethylsulfate and the less expensive DBU as base. Every step of the procedure, amine activation by an o-nitrobenzenesulfonyl group, selective N-methylation and removal of the sulfonamide group, was optimized in respect of time and economy. The described optimized three-step procedure is performed in 35 min without solvent changes, instead of 3 h. Tripeptides (Fmoc-Phe-MeXaa-Leu-OH) containing N-methylated common amino acids were also prepared using the optimized procedure to demonstrate its compatibility with these amino acids. The described procedure allows an efficient synthesis of N(alpha)-methylamino acid containing peptides in a very short time using Fmoc solid-phase peptide synthesis.     

Application of Dmb-Dipeptides in the Fmoc SPPS of Difficult and Aspartimide-Prone Sequences

Mutter’s pseudoproline dipeptides and Sheppard’s Hmb derivatives are powerful tools for enhancing synthetic efficiency in Fmoc SPPS. They work by exploiting the natural propensity of N-alkyl amino acids to disrupt the formation of the secondary structures during peptide assembly. Their use results in better and more predictable acylation and deprotection kinetics, enhanced reaction rates, and improved yields of crude products. However, these approaches have certain limitations: pseudoproline dipeptides can only be used for sequences containing serine or threonine, and the coupling of the amino acid following the Hmb residue can be extremely difficult. To alleviate some of these shortcomings, we have prepared a range of Fmoc-Aaa-(Dmb)Gly-OH dipeptides and tested their efficacy in the synthesis of a number of challenging hydrophobic peptides. We also compared the efficiency of N-Dmb against N-Hmb backbone protection in preventing aspartimide formation in the Fmoc SPPS of peptides containing the Asp-Gly sequence.     

Microwave-assisted Boc-solid phase peptide synthesis of cyclic cysteine-rich peptides.

In this study we describe the first protocols for the synthesis of cystine-rich peptides in the presence of microwave radiation with Boc-solid phase peptide synthesis (SPPS). This method is exemplified for macrocyclic peptides known as cyclotides, which comprise approximately 30 amino acids and incorporate a cystine knot arrangement of their three disulfide bonds. However, the method is broadly applicable for a wide range of peptides using Boc-SPPS, especially for SPPS of large peptides via native chemical ligation. Microwave radiation produces peptides in high yield and with high purity, and we were able to reduce the time for the assembly of approximately 30 mer peptide chains to an overnight reaction in the automated microwave-assisted synthesis.     

Making Ends Meet: Microwave-Accelerated Synthesis of Cyclic and Disulfide Rich Proteins Via In Situ Thioesterification and Native Chemical Ligation

The development of synthetic methodologies for cyclic peptides is driven by the discovery of cyclic peptide drug scaffolds such as the plant-derived cyclotides, sunflower trypsin inhibitor 1 (SFTI-1) and the development of cyclized conotoxins. Currently, the native chemical ligation reaction between an N-terminal cysteine and C-terminal thioester group remains the most robust method to obtain a head-to-tail cyclized peptide. Peptidyl thioesters are effectively generated by Boc SPPS. However, their generation is challenging using Fmoc SPPS because thioester linkers are not stable to repeated piperidine exposure during deprotection. Herein we describe a Fmoc-based protocol for synthesizing cyclic peptides adapted for microwave assisted solid phase peptide synthesis. The protocol relies on the linker Di-Fmoc-3,4-diaminobenzoic acid, and we demonstrate the use of Gly, Ser, Arg and Ile as C-terminal amino acids (using HBTU and HATU as coupling reagents). Following synthesis, an N-acylurea moiety is generated at the C-terminal of the peptide; the resin bound acylurea peptide is then deprotected and cleaved from the resin. The fully deprotected peptide undergoes thiolysis in aqueous buffer, generating the thioester in situ. Ultimately, the head-to-tail cyclized peptide is obtained via native chemical ligation. Two naturally occurring cyclic peptides, the prototypical Möbius cyclotide kalata B1 and SFTI-1 were synthesized efficiently, avoiding potential branching at the diamino linker, using the optimized protocol. In addition, we demonstrate the possibility to use the approach for the synthesis of long and synthetically challenging linear sequences, by the ligation of two truncated fragments of a 50-residue long plant defensin.     

Fluorescein-based amino acids for solid phase synthesis of fluorogenic protease substrates

An efficient synthesis of new type fluorescent amino acids is described. The Fmoc-protected dyes can be prepared in a four-step procedure with approximately 30% overall yield from aminofluoresceins and other inexpensive commercially available precursors. The dyes are much more photostable compared to fluorescein and exhibit constant pH-independent fluorescence that is advantageous in biological applications. The Fmoc-protected fluorescent amino acids are ready for use in solid phase peptide synthesis. As a proof of concept, a fluorogenic papain substrate was synthesized and employed for on-bead detection of the protease activity. By using a novel technique for quantitative analysis of bead fluorescence, a approximately 2.7-fold increase in mean bead brightness was measured and was attributed to substrate cleavage by papain. The new type fluorescent amino acids seem to be a promising tool for the synthesis of fluorescent peptide ligands and fluorogenic protease substrates.     

New method for peptide purification based on selective removal of truncation peptide impurities after SPPS with orthogonal capping

Peptide purification by high-performance liquid chromatography (HPLC) is associated with high solvent consumption, relatively large effort and lack of efficient parallelization. As an alternative, many catch-and-release (c&r) purification methods have been developed over the last decades to enable the efficient parallel purification of peptides originating from solid-phase peptide synthesis (SPPS). However, with one exception, none of the c&r systems has been widely established in industry and academia until today. Herein, we present an entirely new chromatography-free purification concept for peptides synthesized on a solid support, termed reactive capping purification (RCP). The RCP method relies on the capping of truncation peptides arising from incomplete coupling of amino acids during SPPS with a reactive tag. The reactive tag contains a masked functionality that, upon liberation during cleavage from the resin, enables straightforward purification of the peptide by incubation with a resin-bound reactive moiety. In this work, two different reactive tags based on masked thiols were developed. Capping with these reactive tags during SPPS led to effective modification of truncated sequences and subsequent removal of the latter by chemoselective reaction with a maleimide-functionalized solid support. By introducing a suitable protecting group strategy, the thiol-based RCP method described here could also be successfully applied to a thiol-containing peptide. Finally, the purification of a 15-meric peptide by the RCP method was demonstrated. The developed method has low solvent consumption, has the potential for efficient parallelization, uses readily available reagents, and is experimentally simple to perform.     

Synthesis of “Difficult” Fluorescence Quenched Substrates of Granzyme C

The synthesis of fluorescence quenched peptide substrates of granzyme C is presented. These peptides which incorporate some unusual amino acids and have “difficult sequence” elements, in some cases could not be prepared by standard Fmoc-based SPPS. Application of three different contemporary strategies, namely the use of pseudoproline dipeptides, PEG-based solid supports and the application of microwave heating were able to provide for successful synthesis of our desired substrate peptides.     

Pyruvate aldolases in chiral carbon-carbon bond formation

A procedure for the preparation of optically pure alpha-keto-gamma-hydroxy carboxylic acids through stereospecific aldol addition catalyzed by pyruvate aldolases from the Entner-Doudoroff and the DeLey-Doudoroff glycolytic pathways is described. This highly versatile fragment serves as a precursor for a variety of commonly encountered functionalities, including beta-hydroxy aldehydes and carboxylic acids, alpha-amino-gamma-hydroxy carboxylic acids and alpha,gamma-dihydroxy carboxylic acids. The protocol described here uses recombinant His6-tagged KDPG aldolase for the synthesis of (S)-4-hydroxy-2-keto-4-(2'-pyridyl)butyrate. A protocol for evaluating enantiomeric excess through formation of the gamma-lactone of the dithioacetal followed by chiral-phase gas-liquid chromatography is also described. Enzyme expression and enzymatic synthesis can be accomplished in approximately 1 week. The enzymatic aldol addition proceeds in nearly quantitative yields with enantiomeric excesses greater than 99.7%.     

Influenza A virus protein PB1-F2: synthesis and characterization of the biologically active full length protein and related peptides.

Recently the discovery of a novel 87 amino acid influenza A virus (IAV) protein, named PB1-F2, has been reported that originates from an alternative reading frame in the PB1 polymerase gene and is encoded in most of the known human IAV isolates. Using optimized protocols, full length biologically active sPB1-F2 and a number of fragments have been synthesized by following either the standard elongation SPPS method or by native chemical ligation of unprotected N- and C-terminal peptide fragments at the histidine and cysteine residues located in position 41 and 42 of the native sequence, respectively. The ligation procedure afforded the most efficient synthesis of sPB1-F2 and facilitated the generation of various mutants of sPB1-F2 from pre-synthesized peptide fragments. During the synthesis of sPB1-F2, the formation of succinimide and subsequent conversion to the piperidine derivative at the aspartic acid residue in position 23 was observed. This reaction was forestalled by applying specific modifications to the SPPS protocol. The chain-elongation SPPS protocol is optimal for producing small peptides of sPB1-F2, their derivatives and precursors for a subsequent ligation protocol, while the full length protein, mutants and labelled derivatives are more conveniently and efficiently synthesized by SPPS protocols that include native chemical ligation. The molecular identity of sPB1-F2 was confirmed by peptide mapping, mass spectrometry, N-terminal sequencing, (1)H NMR spectroscopy and Western blot analysis. The latter analysis afforded direct evidence of the inherent tendency of sPB1-F2 to undergo oligomerization, a phenomenon observed both for full length sPB1-F2 and fragments thereof, as well as for its full length viral counterpart. Our synthesis protocols open the field for multiple biological and structural studies on sPB1-F2 that, similar to the molecule expressed in an IAV context, induces apoptosis and interacts with membranes in vitro and in vivo, as shown in previous studies.     

Cytophobic surface modification of microfluidic arrays for in situ parallel peptide synthesis and cell adhesion assays

A combination of PEG-based surface passivation techniques and spatially addressable SPPS (solid-phase peptide synthesis) was used to demonstrate a highly specific cell-peptide adhesion assay on a microfluidic platform. The surface of a silicon-glass microchip was modified to form a mixed self-assembled monolayer that presented PEG moieties interspersed with reactive amino terminals. The PEG provided biomolecular inertness and the reactive amino groups were used for consequent peptide synthesis. The cytophobicity of the surface was characterized by on-chip fluorescent binding assays and was found to be resistant to nonspecific attachment of cells and proteins. An integrated system for parallel peptide synthesis on this reactive amino surface was developed using photogenerated acid chemistry and digital microlithography. A constant synthesis efficiency of >98% was observed for up to 7mer peptides. To demonstrate specific cell adhesion on these synthetic peptide arrays, variations of a 7mer cell binding peptide that binds to murine B lymphoma cells were synthesized. Sequence-specific binding was observed on incubation with fluorescently labeled, intact murine B lymphoma cells, and key residues for binding were identified by deletional analysis.     

Solid-phase synthesis of C-terminally modified peptides.

In this paper, a straightforward and generic protocol is presented to label the C-terminus of a peptide with any desired moiety that is functionalized with a primary amine. Amine-functional molecules included are polymers (useful for hybrid polymers), long alkyl chains (used in peptide amphiphiles and stabilization of peptides), propargyl amine and azido propyl-amine (desirable for 'click' chemistry), dansyl amine (fluorescent labeling of peptides) and crown ethers (peptide switches/hybrids). In the first part of the procedure, the primary amine is attached to an aldehyde-functional resin via reductive amination. To the secondary amine that is produced, an amino acid sequence is coupled via a standard solid-phase peptide synthesis protocol. Since one procedure can be applied for any given amine-functional moiety, a robust method for C-terminal peptide labeling is obtained.     

Chemical Synthesis of Maxadilan, a Non-mammalian Potent Vasodilatory Peptide Consisting of 61 Amino Acids with Two Disulfide Bridges, and Its Related Peptides

A potent and persistent non-mammalian derived vasodilator, maxadilan (Maxa) consists of 61 amino acids with two disulfide linkages and acts as an agonist of the type I receptor of pituitary adenylate cyclase activating polypeptide (PACAP), although there is very little sequence similarity. The total chemical syntheses of Maxa, its disulfide isomers and various fragments have been performed successfully by highly efficient solid-phase peptide synthesis (SPPS). A “difficult sequence”, envisaged in the middle region of Maxa, could be overcome by improved synthesis protocols. After assembly peptides were liberated from the resin by cleavage. Peptides having disulfide(s) were purified by two steps of preparative HPLC using cation exchange followed by reverse phase columns. Purified peptides were characterized by HPLC, Edman-sequencing, amino acid analysis and mass spectrometry in addition to disulfide form determination. The peptides obtained were used for recognition studies by the melanophore assay to confirm the native disulfide form. Peptide libraries related to Maxa, produced in the present study, will be useful for the elucidation of the structural requirements of Maxa for interaction with the PACAP type 1 receptor (PAC1). This paper is dedicated to the memory of Professor Bruce Merrifield, a pioneer and one of the most respected experimental scientists, who made extraordinary contributions to high throughput chemical synthesis.     

Efficient synthesis of pyrenylalanine

An efficient synthesis of L-3-(1'-pyrenyl)alanine (Pya), a highly fluorescent amino acid, is described. The amino acid was obtained by the classical asymmetric hydrogenation of chiral 1-acetyl-3-pyrenemethylidene-6-methyl-piperazine-2,5-dione. In the proposed improved procedure mild conditions of the synthesis were applied and the final product--N-tertbutoxycarbonyl-pyrenylalanine--was obtained in good yield. Pyrenylalanine, due to its interesting photophysical properties, can be applied as a fluorescent probe in numerous biochemical and conformational studies.     

Codon-based mutagenesis using dimer-phosphoramidites.

A new approach for the synthesis of randomized DNA sequences containing the 20 codons corresponding to all natural amino acids is described. The strategy is based on the use of dinucleotide phosphoramidite building blocks within a resin-splitting procedure. Through this protocol, a minimal number of seven dimers is sufficient to encode all 20 natural amino acids. This synthesis procedure is extremely flexible and allows codon usage from different hosts to be accommodated.     

Solid-phase synthesis of an Htc-containingdimer analog of the autophosphorylationsite of pp60src PTK: Effective acylationconditions for Htc residues

Summary The difficulty during SPPS in acylating the secondary amino group of Htc, a locally constrained tyrosine, can be correlated with the steric hindrance of the amino acid or with the conformation of the growing peptide chain. Our experimental data indicate that the availability of the Htc amino group is associated with its steric hindrance rather than a conformational effect of the peptide chain. An optimized solid phase automated protocol for Htc is reported. Under optimal conditions, Fmoc-amino acids with hindered side chains were incorporated in approximately 99% yield using HATU as coupling reagent. Unhindered side chain amino acid acylated the secondary amino group of Htc in good yield under classical HBTU/HOBt coupling conditions. Abbreviations: Abbreviations used for amino acids and the designation of peptides follow the rules of the IUPAC-IUB Commission of Biochemical Nomenclature [J. Biol. Chem., 247 (1972) 977–983]. Amino acid symbols denote thel-configuration where applicable, unless indicated otherwise. The following additional, abbreviations are used     

Synthesis of anhydride precursors of the environment-sensitive fluorophores 4-DMAP and 6-DMN

This protocol describes the synthesis of cyclic anhydride precursors of the environment-sensitive fluorophores 4-dimethylaminophthalimide (4-DMAP) and 6-dimethylaminonaphthalimide (6-DMN). The condensation of these anhydrides with a primary amino group confers on molecules of interest solvatochromic properties. In particular, two strategies for the insertion of the chromophores into peptides are presented in two companion protocols. The anhydride syntheses can be completed on the gram scale in 2 d for the 4-DMAP precursor and 10-15 d for the 6-DMN precursor.     

Peptidomimetics Through Click Chemistry: Synthesis of Novel β-Keto Triazole Acids from <Emphasis Type="Italic">N</Emphasis>-Protected Amino Acids

An efficient procedure for the preparation of azidomethylketones from N-urethane protected amino acids and their application in Cu(I) catalyzed azide-alkyne cycloaddition reaction are described. The synthesis has been carried out under mild conditions with all the commonly used urethane protected (Fmoc, Boc and Z) amino acids and the desired azides/triazoles were obtained in good yields. Incorporation of these triazole amino acids into small peptides generating dipeptidomimetics containing β-keto triazole units has also been demonstrated.