All posts in Raw-Journal Peptide Science

Peptide cyclization via chemoselective reactions between side chains has proven a useful strategy to control folded structure. We report here a method for the synthesis of side-chain to side-chain cyclic peptides based on the intermolecular reaction between a linear peptide functionalized with two aminooxy or hydrazide side chains and an organic dialdehyde linker. A family of oxime-based and hydrazone-based cyclic products is prepared in a modular and convergent fashion by combination of unprotected linear peptide precursors and various small molecule linkers in neutral aqueous buffer. The side-chain to side-chain linkages that result can alter peptide folding behavior. The dynamic covalent nature of the Schiff bases in the cyclic products can be utilized to create mixtures where product composition changes in response to experimental conditions. Thus, a linear peptide precursor can select one organic linker from a mixture, and a cyclic product can dynamically exchange the small molecule component of the macrocycle. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.

We report a method for the synthesis of side-chain to side-chain cyclic peptides based on the intermolecular reaction between a linear peptide with two aminooxy or hydrazide side chains and an organic dialdehyde linker. The dynamic covalent nature of the Schiff bases in the cyclic products enables the creation of mixtures where product composition changes in response to experimental conditions.

Here, we report the development of a method for three-fragment assemblies of semisynthetic proteins by combining sortase-mediated ligation with site-specific bioconjugation catalyzed by the 4′-phosphopantetheine transferase Sfp. This method enables the introduction of synthetic peptides into central regions of proteins without the need to purify intermediates. The assembled proteins are linked at the N-terminal junction with a 4′-phosphopantetheine moiety and with a peptide bond at the C-terminal ligation site. We have demonstrated the applicability of this method by assembling a semisynthetic model protein derived from fluorescence resonance energy transfer-based reporters from three fragments in a one-pot reaction. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.

Sortase-mediated ligation in combination with site-specific bioconjugation catalyzed by the 4′-phosphopantetheine transferase Sfp was used for the orthogonal assembly of semisynthetic proteins from three fragments in a one-pot reaction. The assembled proteins are linked at the N-terminal junction with a 4′-phosphopantetheine moiety and with a peptide bond at the C-terminal ligation site. This method was established for model proteins derived from fluorescence resonance energy transfer reporters.

Several naturally occurring peptides in bovine milk were characterized by tandem mass spectrometry and Edman degradation. Chromatograms of peptide fractions (passed through an ultra-filtration membrane, nominal molecular weight limit 3000) prepared from colostrum (collected immediately after parturition) and transitional milk (collected 5 days postpartum) showed that they were almost identical. In total, six peptides, α_s1-CN (f16-23) (RPKHPIKH), α_s1-CN (f16-24) (RPKHPIKHQ), α_s1-CN (f17-25) (PKHPIKHQG), α_s1-CN (f46-52) (VFGKEKV), α_s1-CN (f94-105) (HIQKEDVPSER), and β-CN (f121-128) (HKEMPFPK), were identified. One of the major peptides, the N-terminal fragment of α_s1-casein, varied structurally during early lactation: α_s1-CN (f17-25) (PKHPIKHQG) and α_s1-CN (f16-23) (RPKHPIKH)/α_s1-CN (f16-24) (RPKHPIKHQ) were found in colostrum and transitional milk, respectively. A chemically synthesized peptide, α_s1-CN (f16-23) (RPKHPIKH), inhibited apoptosis of bovine granulosa cells induced by serum-free conditions in a dose-dependent manner, in consequence of caspase-3 and caspase-9 suppressions. The physiological function of the peptide remains unclear, but it may have potential use as pharmaceutical agent and as an anti-apoptotic agent in cell culture medium. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.

An α_s1-casein derived peptide, α_s1-CN (f16-23) (RPKHPIKH), was found in bovine raw milk. Its chemically synthesized peptide inhibited apoptosis of bovine granulosa cells induced by serum-free conditions in a dose-dependent manner.

Synthetic peptide octarphin (TPLVTLFK, a selective agonist of nonopioid β-endorphin receptor) was able to activate in a dose-dependent manner murine macrophages to express nitric oxide (NO) synthase and to produce NO. Octarphin required lipopolysacharide for the optimal induction of NO production. Octarphin-dependent NO production was sensitive to inhibition by dexamethasone and the NO synthase specific inhibitor NG-monomethyl-l-arginine. In the concentration range of 1–1000 nM, octarphin increased the cyclic 3′,5′-guanosine monophosphate (cGMP) content in macrophages stimulated with lipopolysacharide. The effect was dependent on the peptide concentration and was maximal at a concentration of 100 nM. Thus, octarphin stimulates both NO and cGMP production in macrophages. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.

Synthetic peptide octarphin (TPLVTLFK, a selective agonist of nonopioid beta-endorphin receptor) was able to activate in a dose-dependent manner murine macrophages to express nitric oxide (NO) synthase and to produce NO.

Native chemical ligation (NCL) is a powerful method for the convergent synthesis of proteins and peptides. In its original format, NCL between a peptide containing a C-terminal thioester and another peptide offering an N-terminal cysteine has been used to enable protein synthesis of unprotected peptide fragments. However, the applications of NCL extend beyond the scope of protein synthesis. For instance, NCL can be put under the control of template molecules. In such a scenario, NCL enables the design of conditional reaction systems in which, peptide bond formation occurs only when a specific third party molecule is present. In this review, we will show how templates can be used to control the reactivity and chemoselectivity of NCL reactions. We highlight peptide and nucleic-acid-templated NCL reactions and discuss potential applications in nucleic acid diagnosis, origin-of-life studies and gene-expression-specific therapies. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.

Templated chemistry enables the design of reactions, which are under the control of third party molecules. This review describes native chemical ligation reactions that are explored as tools in nucleic acid diagnosis, origin-of-life studies and gene-expression-specific therapies.

Boc-solid phase peptide synthesis is useful for the preparation of peptide--thioesters. However, this strategy usually employs hydrogen fluoride for the final deprotection step. These strongly acidic conditions cannot be applied for the synthesis of acid-labile glycopeptide--thioesters. The protocol presented here is a modified in situ neutralization Boc-solid phase peptide synthesis employing 10% sulfuric acid/dioxane conditions for intermediate Boc removal and TfOH for the final side-chain deprotection step. These conditions were found to be applicable for the synthesis of acid-labile glycopeptide--thioesters. In this protocol, a glycopeptide is synthesized as thioester on a thiol linker, and the product glycopeptide--thioester is released from the resin by thiolysis after side-chain deprotection step with an acid cocktail containing TfOH instead of hydrogen fluoride. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.

The glycopeptide-thioesters are the key components for chemical synthesis of glycoproteins. We report a general procedure for the preparation of glycopeptide-thioesters employing a safe and efficient Boc-solid phase peptide synthesis using 10% sulfuric acid/dioxane for Boc deprotection, low-acidity TfOH conditions for global deprotection, and thiolysis for release of the thioester from the resin.

Negative phototaxis in Archaea is mediated by the sensory rhodopsin II/transducer complex (NpSRII/NpHtrII). After light excitation, the signal is relayed from the receptor to NpHtrII where a rotary motion of TM2 in the membrane domain (NpHtrII_1–114) is induced. This conformational change is transferred to the downstream two-component signaling cascade. Here, we describe the chemical synthesis of this membrane domain, which consists of the two transmembrane helices TM1 and TM2. NpHtrII_1–114 was synthesized using two sequential ligation steps. The first ligation between NpHtrII_47–59 and NpHtrII_60–114 was performed in organic solvents, whereas the final ligation was successful in an aqueous buffer that contained a detergent and a denaturant. The product was refolded into micelles and showed functional properties as determined by binding studies to its cognate receptor NpSRII and by photocycle experiments. This work demonstrates that membrane proteins can be successfully synthesized by chemical means paving the way for tailor-made modifications. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.

We developed a protocol for the synthesis of a membrane protein domain analogue containing two transmembrane helices. The product was refolded into micelles and showed functional properties as determined by binding studies to its cognate receptor NpSRII and by photocycle experiments. This work demonstrates that membrane proteins can be successfully synthesized by chemical means paving the way for tailor-made modifications.

Fluorescence resonance energy transfer (FRET) is a valuable tool for studying protein structure, folding and interactions. The steep distance dependence of the FRET efficiency requires the donor and acceptor to be in close proximity (1–7.5 nm) to exhibit sufficient energy transfer. One possibility to overcome this limitation is the usage of a FRET cascade that utilizes more than one FRET pair. Essential for realizing this FRET cascade is the site-specific introduction of multiple fluorophores to a given protein, which remains a great challenge. In this study, orthogonal labeling techniques, including fluorescent protein tagging, oxime ligation and kinetically controlled cysteine conjugation, are employed to introduce three fluorophores at specific sites of Rab1b GTPase, yielding a triple-labeled FRET probe. The generated protein probe exhibits efficient energy transfer from the primary donor enhanced green fluorescent protein over the intermediate acceptor rhodamine to the final acceptor Dy630. The labeling strategy opens up a new avenue for multi-color labeling of proteins, facilitating long-distance FRET studies. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.

We describe a method for site-specific labeling of a given protein with three distinct fluorophores. This strategy facilitates triple FRET analysis on proteins.

Although proteases are capable of synthesizing peptide bonds via the reverse of proteolysis, they are not proficient at peptide fragment ligation. Further manipulations are needed to shift the native enzyme activity from the cleavage to the synthesis of peptides especially when longer peptides or even proteins are the target molecules of the reaction. This account reports on the synthetic potential of trypsin variants with engineered oxyanion holes mutated by proline mutations, which were designed to minimize proteolytic side reactions during peptide bond synthesis. From the six single and double proline-mutated trypsins, in particular, trypsinQ192P came out as the most promising biocatalyst enabling not only the ligation of cleavage-sensitive peptide fragments but also the selective N-terminal modification of a real protein substrate. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.

For optimizing trypsin for peptide and protein synthesis, the oxyanion hole of the enzyme was engineered by rational mutagenesis. From the six mutated trypsin variants, two enzyme hits with an artificial proline mutation either in position 192 or in position 194 could be identified. Model reactions qualify both biocatalysts as efficient coupling enzymes not only for peptide but also for protein semisynthesis.

A new microbial cyclic dipeptide (diketopiperazine), cyclo(d-Tyr-d-Phe) was isolated for the first time from the ethyl acetate extract of fermented modified nutrient broth of Bacillus sp. N strain associated with rhabditid Entomopathogenic nematode. Antibacterial activity of the compound was determined by minimum inhibitory concentration and agar disc diffusion method against medically important bacteria and the compound recorded significant antibacterial against test bacteria. Highest activity was recorded against Staphylococcus epidermis (1 µg/ml) followed by Proteus mirabilis (2 µg/ml). The activity of cyclo(d-Tyr-d-Phe) against S. epidermis is better than chloramphenicol, the standard antibiotics. Cyclo(d-Tyr-d-Phe) recorded significant antitumor activity against A549 cells (IC₅₀ value: 10 μM) and this compound recorded no cytotoxicity against factor signaling normal fibroblast cells up to 100 μM. Cyclo(d-Tyr-d-Phe) induced significant morphological changes and DNA fragmentation associated with apoptosis in A549 cells. Acridine orange/ethidium bromide stained cells indicated apoptosis induction by cyclo(d-Tyr-d-Phe). Flow cytometry analysis showed that the cyclo(d-Tyr-d-Phe) did not induce cell cycle arrest. Effector molecule of apoptosis such as caspase-3 was found activated in treated cells, suggesting apoptosis as the main mode of cell death. Antioxidant activity was evaluated by free radical scavenging and reducing power activity, and the compound recorded significant antioxidant activity. The free radical scavenging activity of cyclo(d-Tyr-d-Phe) is almost equal to that of butylated hydroxyanisole, the standard antioxidant agent. We also compared the biological activity of natural cyclo(d-Tyr-d-Phe) with synthetic cyclo(d-Tyr-d-Phe) and cyclo(l-Tyr-l-Phe). Natural and synthetic cyclo(d-Tyr-d-Phe) recorded similar pattern of activity. Although synthetic cyclo(l-Tyr-l-Phe) recorded lower activity. But in the case of reducing power activity, synthetic cyclo(l-Tyr-l-Phe) recorded significant activity than natural and synthetic cyclo(d-Tyr-d-Phe). The results of the present study reveals that cyclo(d-Tyr-d-Phe) is more bioactive than cyclo(l-Tyr-l-Phe). To the best of our knowledge, this is the first time that cyclo(d-Tyr-d-Phe) has been isolated from microbial natural source and also the antibacterial, anticancer, and antioxidant activity of cyclo(d-Tyr-d-Phe) is also reported for the first time. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.

A new microbial cyclic dipeptide (diketopiperazine), cyclo(D-Tyr-D-Phe) was isolated for the first time from the ethyl acetate extract of fermented modified nutrient broth of Bacillus sp. N strain associated with rhabditid entomopathogenic nematode. The antibacterial, anticancer and antioxidant activity of cyclo(d-Tyr-d-Phe) is also reported for the first time. We also compared the biological activity of natural cyclo(d-Tyr-d-Phe) with synthetic cyclo(d-Tyr-D-Phe) and cyclo(L-Tyr-L-Phe). Natural and synthetic cyclo(d-Tyr-d-Phe) recorded similar pattern of activity, whereas synthetic cyclo(L-Tyr-L-Phe) recorded lower activity. This clearly indicated that stereochemistry plays an important role in the bioactivity of cyclic dipeptides.