Dehydroalanine-containing peptides: preparation from phenylselenocysteine and utility in convergent ligation strategies

This protocol describes the methodology for the synthesis of dehydroalanine (Dha)-containing peptides and illustrates their use in convergent ligation strategies for the preparation of peptide conjugates. A nonproteinogenic amino acid, Fmoc-Se-phenylselenocysteine (SecPh), can be prepared in high yield over four synthetic steps and be conveniently incorporated into peptides by standard solid-phase peptide synthesis techniques. Globally deprotected peptides containing phenylselenocysteine can be converted to dehydrated peptides following a chemoselective, mild oxidation with hydrogen peroxide or sodium periodate (i.e., the phenylselenocysteine side chain is converted to that of Dha). Dha residues are electrophilic handles for the preparation of glycopeptides, lipopeptides or other peptide conjugates; one such transformation will be outlined here. The preparation of Dha-containing peptides, including the synthesis of SecPh, peptide elongation and oxidative treatment of phenylselenocysteine-containing peptides can be completed by one person in approximately 3-5 weeks. However, once SecPh is in hand, the time required for the preparation of peptides is significantly shorter and comparable to that for any peptide synthesis.     

A peptidyl transferase ribozyme capable of combinatorial peptide synthesis

The formation of peptide bonds is a key step in both the chemical and biological synthesis of peptides. The ribozyme can use a wide range of amino acids as its substrate for the dipeptide synthesis. A library containing 29 peptides whose synthesis was catalyzed by this unique ribozyme was analyzed by mass spectrometry. These results implicate that ribozyme may have potential application in the peptide synthesis.     

Derivatization of peptides as quaternary ammonium salts for sensitive detection by ESI‐MS

A series of model peptides in the form of quaternary ammonium salts at the N‐terminus was efficiently prepared by the solid‐phase synthesis. Tandem mass spectrometric analysis of the peptide quaternary ammonium derivatives was shown to provide sequence confirmation and enhanced detection. We designed the 2‐(1,4‐diazabicyclo[2.2.2] octylammonium)acetyl quaternary ammonium group which does not suffer from neutral losses during MS/MS experiments. The presented quaternization of 1,4‐diazabicyclo[2.2.2]octane (DABCO) by iodoacetylated peptides is relatively easy and compatible with standard solid‐phase peptide synthesis. This methodology offers a novel sensitive approach to analyze peptides and other compounds. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

A Dde resin based strategy for inverse solid-phase synthesis of amino terminated peptides, peptide mimetics and protected peptide intermediates.

This report describes a Dde resin based attachment strategy for inverse solid-phase peptide synthesis (ISPPS). This attachment strategy can be used for the synthesis of amino terminated peptides with side chains and the carboxyl terminus either protected or deprotected. Amino acid t-butyl esters were attached through their free amino group to the Dde resin. The t-butyl carboxyl protecting group was removed by 50% TFA, and inverse peptide synthesis cycles performed using an HATU/TMP based coupling method. Protected peptides were cleaved from the resin with dilute hydrazine. Side chain protecting groups could then be removed by treatment with TFMSA/TFA. The potential of this approach was demonstrated by the synthesis of several short protected and unprotected peptides in good yield and with low epimerization. Its potential for peptide mimetic synthesis was demonstrated by the synthesis of two peptide trifluoromethylketones.     

Monitoring enzyme synthesis as a means of studying peptide transport and utilization in Escherichia coli.

A new method has been developed for measuring peptide transport in aminoacid auxotrophs of Escherichia coli by following induction of beta-galactosidase. Appearance of the enzyme was determined after addition of inducer and peptides to amino-acid starved bacteria. For a given number of lysine equivalents, the rate and the extent of enzyme synthesis were the same for lysine and lysyl peptides; similar results were found for glycine and glycl peptides. Saturation constants for peptide transport were determined from the exogenous peptide concentration that gave half maximal rates of enzyme synthesis. The saturation constants, studies with mutants defective in peptide transport, and detection of competition between peptides for uptake, all endorsed earlier conclusions from growth tests about the structural specificities for peptide transport. The new method is quicker, more sensitive and more informative than growth tests.     

Multiple peptide synthesis

The synthesis of large numbers of peptides can be very labor intensive and, if a conventional peptide synthesizer is used, only small numbers of peptides can be produced within a reasonable time. The techniques described below can make large numbers of different peptides simultaneously with varying degrees of mechanization, ranging from the wholly manual methods, to those involving complete mechanization of the whole synthesis process. Most of the multiple synthesis methods are primarily intended for small scale production ranging from microgram amounts up to a few tens of milligrams. All of the systems are economical in use of solvents and reagents, enabling cost-effective synthesis. The techniques described can also be used to prepare peptide libraries, containing several millions of peptide sequences, to enable the rapid screening of all possible permutations of amino acids within short peptides. However, it is considered that multiple synthesis methods are not particularly suited where extreme high purity or very long peptides are required.     

Amino acid pyridoxyl esters in peptide synthesis

Pyridoxyl residue was suggested to be used as a multifunctional protective and modifying group in peptide synthesis. The modification was carried out by introducing the pyridoxyl residue in free or partially protected peptides or by the addition of amino acid pyridoxyl esters by the methods of conventional peptide synthesis without the removal of the pyridoxyl group at the terminal stages of the synthesis (the second approach is more convenient). Pyridoxyl residue was also used as a spacer in solid phase peptide synthesis. It was attached to the polymer by the alkylation of the hydroxyl groups or of the pyridine ring of the pyridoxyl derivatives with the chloromethylated styrene-divinylbenzene copolymer (the standard Merrifield resin). Potentials for the use of pyridoxyl derivatives in the synthesis of linear, multiplet, and cyclic peptides are discussed.     

Convergent synthesis of aminomethylene peptidomimetics

This protocol describes a convergent synthesis of reduced amide bond peptidomimetics using thioacid-terminated peptides and aziridine-containing peptide conjugates. This approach could be used to produce peptides and proteins with modified backbones. The peptide conjugates are made using readily available aziridine aldehydes. The convergent synthesis of peptidomimetics is demonstrated through the preparation of long and short peptide fragments with an aminomethylene group incorporated within them. This transformation is amenable to the synthesis of peptides with reduced amide bonds at cysteine and alanine. The procedure describes the preparation of each component used and highlights the ease of synthesis of aminomethylene peptidomimetics, and takes about 3 d to complete.     

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.     

Fmoc/solid-phase synthesis of Tyr(P)-containing peptides through t-butyl phosphate protection.

The synthesis of Tyr(P)-containing peptides by the use of Fmoc-Tyr(PO3Me2)-OH in Fmoc/solid phase synthesis is complicated since, firstly, piperidine causes cleavage of the methyl group from the -Tyr(PO3Me2)-residue during peptide synthesis and, secondly, harsh conditions are needed for its final cleavage. A very simple method for the synthesis of Tyr(P)-containing peptides using t-butyl phosphate protection is described. The protected phosphotyrosine derivative, Fmoc-Tyr(PO3tBu2)-OH was prepared in high yield from Fmoc-Tyr-OH by a one-step procedure which employed di-t-butyl N,N-diethyl-phosphoramidite as the phosphorylation reagent. The use of this derivative in Fmoc/solid phase peptide synthesis is demonstrated by the preparation of the Tyr(P)-containing peptides, Ala-Glu-Tyr(P)-Ser-Ala and Ser-Ser-Ser-Tyr(P)-Tyr(P).     

Matrix‐assisted peptide synthesis on nanoparticles

We report a new method for multistep peptide synthesis on polymeric nanoparticles of differing sizes. Polymeric nanoparticles were functionalized via their temporary embedment into a magnetic inorganic matrix that allows multistep peptide synthesis. The matrix is removed at the end of the process for obtaining nanoparticles functionalized with peptides. The matrix‐assisted synthesis on nanoparticles was proved by generating various biologically relevant peptides. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Development of caged non-hydrolyzable phosphoamino acids and application to photo-control of binding affinity of phosphopeptide mimetic to phosphopeptide-recognizing protein

The design and synthesis of caged non-hydrolyzable phospho-serine, -threonine, and -tyrosine derivatives that generate parent non-hydrolyzable phosphoamino acids, containing a difluoromethylene unit instead of the oxygen of a phosphoester, after UV-irradiation are described. The caged non-hydrolyzable amino acids were incorporated into peptides by standard Fmoc solid-phase peptide synthesis, and the obtained peptides were successfully converted to the parent non-hydrolyzable phosphopeptides by UV-irradiation. Application of the caged non-hydrolyzable phosphoserine-containing peptide to photo-control the binding affinity of the peptide to 14-3-3β protein is also reported.     

Use of the multipin peptide synthesis technique for the generation of antipeptide sera

The multipin peptide synthesis technique has been used to map antigenic sites of proteins (1,2). Antibodies raised to the whole protein are screened on pin-synthesized overlapping octapeptides homologous with the protein of interest, and the peptides that bind antibodies clearly identify the epitopes. What is described in this study is a method using pin-synthesized peptides to generate specific antibodies to many peptides. Cleavable linkers have been developed (3) that, used together with the multipin peptide synthesis technique, allow the synthesis and cleavage of many thousands of peptides into aqueous solutions at physiological pH. This technique is useful for assays requiring peptides in solution, e.g., mapping of T-cell determinants. A technique has been developed for the cleavage of many peptides from pins and simultaneous coupling to immunogenic carriers (4). The conjugates produced are suitable for the generation of antipeptide antibodies. This procedure is illustrated using several 15 amino acid long peptides (15-mers), homologous with the sequence of a model antigen, myohemerythrin (MHr). The resulting antipeptide sera generated were tested by ELISA for titer and specificity on pinsynthesized peptides and β-amide peptides and the protein antigen coated to microtiter plates.     

Is crustacean hyperglycaemic hormone precursor-related peptide a circulating neurohormone in crabs?

Sites of synthesis and release patterns of crustacean hyperglycaemic hormone precursor-related peptide (CPRP) were investigated with those of crustacean hyperglycaemic hormone (cHH), in order to determine whether this precursor-related peptide satisfies certain criteria necessary for its definition as a secretable, circulating hormone. Using the edible crab, Cancer pagurus, sites of CPRP synthesis were determined by immunohistochemistry and release patterns of both peptides were determined in vivo and in vitro by radioimmunoassay of haemolymph and eyestalk superfusates. Both peptides were co-released from sinus glands (SGs) following potassium-evoked depolarization of isolated eyestalk preparations. However, stress-evoked in vivo release resulted in apparent non-stoichiometric circulating peptide profiles. This phenomenon is explained by notable differences in clearance rates of the peptides in haemolymph. In contrast to cHH, CPRP is very slowly degraded in vivo. Although CPRP is clearly a circulating peptide, whose release is concomitant with that of cHH, physiologically pertinent roles for this molecule remain to be discovered.     

Synthesis and biological activity of mouse hepcidin peptide analogs containing three disulfide bridges: manual and microwave-assisted solid-phase peptide synthesis

Hepcidin, a 25 amino acid peptide hormone containing a complex network of four disulfide bonds is the hormone regulator of iron homeostasis. Three bridges synthetic peptide analogs have been prepared following two synthetic strategies and two oxidation procedures: i) a microwave-assisted solid phase synthesis followed by air oxidation of the six free cysteines ii) a manual solid phase synthesis followed by stepwise deprotection and oxidation of cysteine pairs. All the peptides with different connectivities have been characterized by MALDI ToF spectrometry, and tested for their ability to degrade the cellular iron exporter, ferroportin. While linear peptides are inactive, the one-bridge and two-bridge peptides retaining protected cysteines by bulky substituents are active. Similarly, the three-bridge peptides are active irrespective of their disulfide connectivities.     

In vitro translation of the upstream open reading frame in the mammalian mRNA encoding S-adenosylmethionine decarboxylase

The upstream open reading frame (uORF) in the mRNA encoding S-adenosylmethionine decarboxylase is a polyamine-responsive element that suppresses translation of the associated downstream cistron in vivo. In this paper, we provide the first direct evidence of peptide synthesis from the S-adenosylmethionine decarboxylase uORF using an in vitro translation system. We examine both the influence of cation concentration on peptide synthesis and the effect of altering the uORF sequence on peptide synthesis. Synthesis of wild type and altered peptides was similar at all concentrations of magnesium tested. In contrast, synthesis of the wild type peptide was more sensitive than that of altered peptides to elevated concentrations of the naturally occurring polyamines, spermidine and spermine, as well as several polyamine analogs. The sensitivity of in vitro synthesis to spermidine was influenced by both the amino acid sequence and the length of the peptide product of the uORF. Findings from the present study correlate with the effects of the uORF and polyamines on translation of a downstream cistron in vivo and support the hypothesis that polyamines and the structure of the nascent peptide create a rate-limiting step in uORF translation, perhaps through a ribosome stalling mechanism.     

Chemical synthesis of cell-permeable apoptotic peptides from in vivo produced proteins

In vivo synthesis of peptides by bacterial expression has developed into a reliable alternative to solid-phase peptide synthesis. A significant drawback of in vivo methods is the difficulty with which gene products can be modified post-translationally. Here, we present a method for the facile modification of peptides generated in bacterial hosts after cyanogen bromide cleavage at C-terminal methionines. Reaction of the resulting homoserine lactones with propargylamine allows efficient and selective modification with a wide variety of chemicals such as fluorescent dyes, biotin derivatives, polyprenyls, lipids, polysaccharides, or peptides. Attachment of the cell penetrating peptide octa-arginine (R(8)) to peptides derived from the proapoptotic tumor suppressor Bak BH3 led to efficient cellular uptake and subsequent cytochrome c release from mitochondria, culminating in induction of apoptosis similar to that observed with peptides linked to R(8) via the peptide backbone. These results highlight the significant potential for use of such tools in live cells.     

On the chemical basis of the Lowry protein determination

The copper-catalyzed oxidation of peptides and proteins by phosphomolybdic/phosphotungstic acid (Folin phenol reagent) was studied with respect to redox stoichiometry of color formation and nature of the oxidation products. From peptides without reducing side chains two reducing equivalents were transferred under ideal conditions to Mo6+/W6+ for each unit of tetradentate copper complex with concomitant formation of an imino peptide. Tyrosine and tryptophan side chains contributed four additional reducing equivalents. Oxidation of proline-containing peptides was greatly impaired as judged from color formation due to the interference of the imino acid with complex formation. Reaction of the oxidized peptides with 2,4-dinitrophenyl (DNP)-hydrazine gave a peptide amine and the DNP-hydrazone of a 2-oxoacyl peptide. The oxidation products from tetraalanine were identified as dialanine amide and pyruvoylalanine DNP-hydrazone. From the time course of the development of the blue color on reduction of Folin reagent with tetraalanine it was inferred that the reaction consisted of an initial (less than 5 s) oxidation to a Cu3+ peptide complex followed by slow changes in absorbance, especially above 0.2 mM. Due to these complications the two-electron stoichiometry has to be considered only as a limiting case for peptide concentrations below 0.02 mM.     

Pyridoxyl amino acid esters in peptide synthesis

Pyridoxyl residue was suggested to be used as a multifunctional protective and modifying group in peptide synthesis. The modification was carried out by introducing the pyridoxyl residue in free or partially protected peptides or by the addition of amino acid pyridoxyl esters by the methods of conventional peptide synthesis without the removal of the pyridoxyl group at the terminal stages of the synthesis (the second approach is more convenient). Pyridoxyl residue was also used as a spacer in solid phase peptide synthesis. It was attached to the polymer by the alkylation of the hydroxyl groups or of the pyridine ring of the pyridoxyl derivatives with the chloromethylated styrene-divinylbenzene copolymer (the standard Merrifield resin). Potentials for the use of pyridoxyl derivatives in the synthesis of linear, multiplet, and cyclic peptides are discussed.