Ribosomal synthesis of unnatural peptides

Combinatorial libraries of non-biological polymers and drug-like peptides could in principle be synthesized from unnatural amino acids by exploiting the broad substrate specificity of the ribosome. The ribosomal synthesis of such libraries would allow rare functional molecules to be identified using technologies developed for the in vitro selection of peptides and proteins. Here, we use a reconstituted E. coli translation system to simultaneously re-assign 35 of the 61 sense codons to 12 unnatural amino acid analogues. This reprogrammed genetic code was used to direct the synthesis of a single peptide containing 10 different unnatural amino acids. This system is compatible with mRNA-display, enabling the synthesis of unnatural peptide libraries of 10(14) unique members for the in vitro selection of functional unnatural molecules. We also show that the chemical space sampled by these libraries can be expanded using mutant aminoacyl-tRNA synthetases for the incorporation of additional unnatural amino acids or by the specific posttranslational chemical derivitization of reactive groups with small molecules. This system represents a first step toward a platform for the synthesis by enzymatic tRNA aminoacylation and ribosomal translation of cyclic peptides comprised of unnatural amino acids that are similar to the nonribosomal peptides.     

An Optimized Protocol for the Synthesis of Peptides Containing trans-Cyclooctene and Bicyclononyne Dienophiles as Useful Multifunctional Bioorthogonal Probes

Despite the great advances in solid-phase peptide synthesis (SPPS), the incorporation of certain functional groups into peptide sequences is restricted by the compatibility of the building blocks with conditions used during SPPS. In particular, the introduction of highly reactive groups used in modern bioorthogonal reactions into peptides remains elusive. Here, we present an optimized synthetic protocol enabling installation of two strained dienophiles, trans-cyclooctene (TCO) and bicyclononyne (BCN), into different peptide sequences. The two groups enable fast and modular post-synthetic functionalization of peptides, as we demonstrate in preparation of peptide-peptide and peptide-drug conjugates. Due to the excellent biocompatibility, the click-functionalization of the peptides can be performed directly in live cells. We further show that the introduction of both clickable groups into peptides enables construction of smart, multifunctional probes that can streamline complex chemical biology experiments such as visualization and pull-down of metabolically labeled glycoconjugates. The presented strategy will find utility in construction of peptides for diverse applications, where high reactivity, efficiency and biocompatibility of the modification step is critical.     

Peptidyl-Resin Substrates as a Tool in the Analysis of Caspase Activity

Caspases, proteolytic enzymes belonging to the group of cysteine proteases, play a crucial role in apoptosis. Understanding their activity and substrate specificity is extremely important. Fluorescence-based approaches, including fluorogenic substrates, are generally used to confirm cleavage preferences. Here we present a new method of substrate specificity and activity analysis based on the application of fix-charge tagged peptides located on the resin. The proteolysis of peptide bond on the resin, occurring even with low efficiency, results in the formation of N-terminal fragments of model peptide containing ionization enhancers in the form of quaternary ammonium groups, allowing for ultrasensitive and reliable analysis by LC-MS/MS. The possibility of application of the proposed solution was tested through the analysis of substrate specificity and activity of caspase 3 or 7. The obtained results confirm the known substrate specificity of executioner caspases. Our solution also allowed us to observe that caspases can hydrolyze peptides shorter than those presented to date in the scientific literature.     

Construction and activity of a synthetic basement membrane with active laminin peptides and polysaccharides

Cell-adhesive peptides derived from extracellular matrix (ECM) proteins are potential candidates for incorporating cell-binding activities into materials for tissue engineering. We have identified a number of cell adhesive peptides from laminins, which are major components of basement membrane ECM. Our goal is the development of synthetic basement membranes using the peptides on scaffolds. We review peptide–polysaccharide complexes, which were prepared by conjugation of the peptides to chitosan and alginate, and the biological activities of the resulting matrices. The peptide–polysaccharide matrices can also be used as a biomaterial for cell transplantation. These studies suggest that the peptide–polysaccharide complexes have the potential to mimic the multifunctional basement membrane and may be useful for tissue engineering.     

Adducts of N-terminal valines in hemoglobin with isoprene diepoxide, a metabolite of isoprene

Isoprene (2-methylbuta-1,3-diene) is a multi-site carcinogen in rodents. To evaluate the role of the diepoxide metabolite (1,2:3,4-diepoxy-2-methylbutane) in carcinogenesis, measurements of in vivo doses of the diepoxide are needed. The in vivo dose may be inferred from levels of reaction products with hemoglobin (Hb adducts). This report presents in vitro studies of the adduct formation by the diepoxide of isoprene with valinamide and oligopeptides as model compounds of N-terminal valines in hemoglobin (Hb). In the reaction with valinamide it was shown that isoprene diepoxide forms as the main product a ring-closed adduct, which is a pyrrolidine derivative [N,N-(2,3-dihydroxy-2-methyl-1,4-butadiyl)valinamide, MPyr-Val]. The analysis was performed by gas chromatography/mass spectrometry (GC/MS) (EI and PICI) after acetylation. The ring-closed adduct was also identified by liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) as the main product in the reaction between isoprene diepoxide and standard hepta- or (2H8)octapeptides, corresponding to the N-terminal peptides of the alpha-chains in mouse and rat Hb. These peptides, alkylated with isoprene diepoxide, to be used as internal standards and calibration standards for quantification of MPyr-adduct levels in vitro and in vivo, were analyzed with respect to the degree of MPyr-alkylation by two independent methods, amino acid analysis and HPLC-UV; similar results were obtained using these methods. A method for measurement of Hb adducts as modified peptides, used earlier to measure a similar adduct to N-terminal valines in Hb from the diepoxide of 1,3-butadiene, has in the present work been tested for application to isoprene diepoxide. The method is based on tryptic degradation of globin and LC/ESI-MS analysis of N-terminal Pyr-heptapeptides of the Hb alpha-chain enriched by HPLC. MPyr-adduct levels in isoprene diepoxide alkylated hemolysate from mouse erythrocytes incubated with different concentrations of isoprene diepoxide (2 and 10 mM) for 1 h were quantified. The adduct level was about 50 nmol/g alpha-chain Hb per mM x h. From the adduct levels the rate constant of isoprene diepoxide for reaction with N-terminal valine was calculated to be about 1.6 times faster than for diepoxybutane.     

Mass spectrometric measurement of endogenous peptides

Mass spectrometric analytical techniques utilizing fast atom bombardment are used to ionize peptides extracted from biological sources, and tandem mass spectrometry methods are used to select a unique amino acid sequence-determining fragment ion for quantification of that peptide at the picomole level. This type of analysis maximizes the molecular specificity.     

Deviation from the mobile proton model in amino-modified peptides: implications for multiple reaction monitoring analysis of peptides

The study of peptide fragmentation is important to the understanding of chemical processes occurring in the gas phase and the more practical concern of peptide identification for proteomic analysis. Using the mobile proton model as a framework, we explore the effect of amino-group modifications on peptide fragmentation. Three aldehydes are used to transform the peptides' primary amino groups into either a dimethylamino or a heterocyclic structure (five- or six-membered). The observed fragmentation patterns deviate strongly from those observed for the analogous underivatised peptides. In particular, the a1 ion is the base peak in most tandem mass spectra of the derivatised peptides. The a1 ion intensity depends strongly on the N-terminal amino acid, with tyrosine and phenylalanine having the strongest enhancement. Despite the change in fragmentation patterns of the derivatised peptides, they still provide high-quality tandem mass spectra that, in many cases, are more amenable to database searching than the spectra of underivatised peptides. In addition, the reliable presence of the a1 ion facilitates rapid quantitative measurements using the multiple reaction monitoring approach.     

The oxime bond formation as an efficient tool for the conjugation of ruthenium complexes to oligonucleotides and peptides

A convenient method for the conjugation of ruthenium complex on oligonucleotides and peptides through chemoselective oxime linkage is reported. Novel Ru(II) complexes sustaining an aminooxy containing ligand were prepared and efficiently coupled with the oligonucleotides and peptides functionalized with the complementary reactive aldehyde group. The method described herein could be a useful tool for preparing a broad range of metal complex-oligonucleotide and peptide conjugates.     

Selective Alkaline Protease Catalyzed Hydrolysis of Peptide Esters

Procedures for preparing C-terminal free peptides from hydrolysis of its corresponding methyl or benzyl esters catalyzed by alkaline protease has been developed. N-protected peptides having side-chain ester protecting groups or successive hydrophobic amino acid residues in its sequence are hydrolyzed selectively at C-terminal only and leave other bonds (β and γ- ester or peptide bonds) intact. Compounds which cause a side reaction in base mediated saponification could be hydrolyzed safely by this procedure. Products of this hydrolysis are useful intermediates for fragments coupling in the solid phase peptide synthesis.     

Recognition of peptides by antibodies and investigations of affinity using biosensor technology

The study of peptide-antibody interactions has many applications in biology and medicine. Synthetic peptides corresponding to single protein epitopes are used instead of intact proteins as reagents for the diagnosis of viral and autoimmune diseases. Furthermore, antibodies raised against peptides are useful reagents for isolating and characterizing gene products. In this review, methods for analysing the molecular basis of peptide-antibody interactions are described, such as amino acid replacement studies, X-ray crystallography of peptide-antibody complexes and biosensor technology based on surface plasmon resonance. The importance of peptide conformation in antibody recognition is discussed, and the antigenic reactivity of epitopes in synthetic peptides and in cognate, intact proteins is compared.     

Biomimetic studies on the mechanism of stereoselective lanthionine formation

Selenocysteine derivatives are useful precursors for the synthesis of peptide conjugates and selenopeptides. Several diastereomers of Fmoc-3-methyl-Se-phenylselenocysteine (FmocMeSec(Ph)) were prepared and used in solid phase peptide synthesis (SPPS). Once incorporated into peptides, the phenylselenide functionality provides a useful handle for the site and stereospecific introduction of E- or Z-dehydrobutyrine residues into peptide chains via oxidative elimination. The oxidation conditions are mild, can be performed on a solid support, and tolerate functionalities commonly found in peptides, including variously protected cysteine residues. Dehydropeptides containing unprotected cysteine residues undergo intramolecular stereoselective conjugate addition to afford cyclic lanthionines and methyllanthionines, which have the same stereochemistry as found in lantibiotics, a family of ribosomally synthesized and post-translationally modified peptide antibiotics. The observed stereoselectivity is shown to originate from a kinetic rather than a thermodynamic preference.     

LC/MS characterization of undesired products formed during iodoacetamide derivatization of sulfhydryl groups of peptides

Many undesired by-products have been noticed during alkylation with iodoacetamide, a widely used derivatization reaction in proteomics for the determination of sulfhydryl groups in peptides and proteins. We report here that iodoacetamide reacts with the N-terminal NH2 and the C-terminal carboxylic acid groups, in addition to the peripheral residues bearing protic functional groups. If sufficient reaction time is given, the N-terminal NH2 group is readily dialkylated by iodoacetamide. In fact, the N-terminal NH2 group reacts even faster than the reactive sites present in residues, such as tyrosine or histidine. LC/MS investigations with certain reactive peptides show that by-products are formed in a relatively short reaction time, even at room temperature. Interestingly, derivatives formed in this way are useful for sequence determination of peptides by MS since the intensities of y' ions are highly suppressed in the spectra of N-terminus mono- and dialkylated peptides, whereas those of b-ions are significantly enhanced. For example, in the spectrum of N,N-dicarboxamidomethyl derivative of Val-Ala-Ala-Phe (VAAF), the y-series ions are virtually absent. On the other hand, when the derivatization takes place at the carboxylic group, the y-series ions are markedly observed in the spectra of these undesired O-carboxamidomethyl derivatives.     

Controlled peptide solvation in portion-mixing libraries of FRET peptides: improved specificity determination for Dengue 2 virus NS2B-NS3 protease and human cathepsin S

The solubility of peptides in aqueous buffers used for the enzyme assays is a common limitation for all peptide libraries. In principle, the more water-soluble peptides are, the more susceptible they will be to peptidase hydrolysis. We have demonstrated that this bias can be circumvented in a portion-mixing fluorescence resonance energy transfer (FRET) peptide library by introducing k (lysine in the D-form) in both termini of the peptides. This more solvated library and another one without the k were assayed using trypsin and chymotrypsin as standard peptidases with high selectivity for R and K and for hydrophobic F and Y, respectively. Significantly improved consistency of the information on substrate profiles was obtained from the solvated library. The influence of improved solvation on substrate specificity determination was successfully demonstrated by the difference in specificity observed between the two libraries employing the human cathepsin S (accepts acidic, basic, or neutral amino acids at P1 position) and Dengue 2 virus NS2B-NS3 protease (high specificity to the pair of basic amino acids K-R, R-R, or Q-R/K at P2-P1 positions). In conclusion, hydration of the peptides has a major influence on protease processing, and this bias can be reduced in bound peptide libraries, improving reliability.     

Intermolecular condensation products formed during the pyrolysis of peptides

Mass Spectrometry (MS) analysis of pyrolysis products of simple peptides has revealed several non-volatile thermal degradation products at masses lower than the precursor peptide. In addition to these products, many other signals were also observed at higher masses than the precursor peptide, and their characterization is the focus of this study. Here we report on the observation of homo and hetero condensation peptide products formed during the pyrolysis of peptides. The observed peptide condensation products are formed between two, three or even four peptides. Tandem MS (MS/MS) analyses of these products showed that C-terminal to N-terminal intermolecular bonding is preferred during pyrolysis when combining two peptides, rather than involving crosslinking between basic and acidic side chain groups like arginine and aspartic acid. These observations are rationalized by steric hindrance effect and known pK_a values of the peptide C- and N-termini and amino acid side groups like aspartic acid and arginine. Pyrolysis of a standard N-acetylated peptide showed no detectable condensation and/or crosslinked products, even in peptides with basic side groups, providing further evidence for the C-terminus to N-terminus intermolecular bonding between peptides under pyrolytic conditions.     

Biologically-Inspired Peptide Reagents for Enhancing IMS-MS Analysis of Carbohydrates

The binding properties of a peptidoglycan recognition protein are translated via combinatorial chemistry into short peptides. Non-adjacent histidine, tyrosine, and arginine residues in the protein’s binding cleft that associate specifically with the glycan moiety of a peptidoglycan substrate are incorporated into linear sequences creating a library of 27 candidate tripeptide reagents (three possible residues permutated across three positions). Upon electrospraying the peptide library and carbohydrate mixtures, some noncovalent complexes are observed. The binding efficiencies of the peptides vary according to their amino acid composition as well as the disaccharide linkage and carbohydrate ring-type. In addition to providing a charge-carrier for the carbohydrate, peptide reagents can also be used to differentiate carbohydrate isomers by ion mobility spectrometry. The utility of these peptide reagents as a means of enhancing ion mobility analysis of carbohydrates is illustrated by examining four glucose-containing disaccharide isomers, including a pair that is not resolved by ion mobility alone. The specificity and stoichiometry of the peptide–carbohydrate complexes are also investigated. Trihistidine demonstrates both suitable binding efficiency and successful resolution of disaccharides isomers, suggesting it may be a useful reagent in IMS analyses of carbohydrates.     

Fabrication of Chiral Materials via Self‐Assembly and Biomineralization of Peptides

With different scales of chirality, chiral materials have various particular properties and potential applications in many fields. Peptides are the fundamental building units of biological systems, and a variety of ordered functional nanostructures are produced through self‐assembly and biomineralization of peptides in nature. This Personal Account describes chiral silica materials fabricated by using amphiphilic peptides as building blocks. Three particular biomineralization approaches are described based on different kinds of geometry of amphiphilic peptides: the influence of the specific amino acid proline in the peptide sequence, the hydrophilicity of amphiphilic peptides, and different kinds of hydrophobic tails in amphiphilic peptides. These strategies are useful for designing peptides toward the bottom‐up synthesis of nanomaterials as well as improving the understanding of the mechanism of peptide self‐assembly.     

De novo Design of Selective Membrane‐Active Peptides by Enzymatic Control of Their Conformational Bias on the Cell Surface

Selectively targeting the membrane‐perturbing potential of peptides towards a distinct cellular phenotype allows one to target distinct populations of cells. We report the de novo design of a new class of peptide whose ability to perturb cellular membranes is coupled to an enzyme‐mediated shift in the folding potential of the peptide into its bioactive conformation. Cells rich in negatively charged surface components that also highly express alkaline phosphatase, for example many cancers, are susceptible to the action of the peptide. The unfolded, inactive peptide is dephosphorylated, shifting its conformational bias towards cell‐surface‐induced folding to form a facially amphiphilic membrane‐active conformer. The fate of the peptide can be further tuned by peptide concentration to affect either lytic or cell‐penetrating properties, which are useful for selective drug delivery. This is a new design strategy to afford peptides that are selective in their membrane‐perturbing activity.     

N-terminus FITC labeling of peptides on solid support: the truth behind the spacer

Fluorescein isothiocyanate (FITC) is an amine reactive derivative of fluorescein dye that has wide ranging application in biochemistry. It has been extensively used to label peptides and proteins. However, its use in solid phase peptide synthesis is restricted. Indeed, in acidic conditions required for linker cleavage, N-ter FITC-labeled peptides undergo a cyclization leading to the formation of a fluorescein with subsequent removal of the last amino acid. This can be avoided when a spacer such as amino hexanoic acid is used or if non-acidic cleavage is operated to release targeted peptide from the resin.     

Cyclic voltammetric study of the redox system of glutathione using the disulfide bond reductant tris(2-carboxyethyl)phosphine

The stabilization of the reduction state of proteins and peptides is very important for the monitoring of protein-protein, protein-DNA and protein-xenobiotic interactions. The reductive state of protein or peptide is characterized by the reactive sulfhydryl group. Glutathione in the reduced (GSH) and oxidized (GSSG) forms was studied by cyclic voltammetry. Tris(2-carboxyethyl)phosphine (TCEP) as the disulfide bond reductant and/or hydrogen peroxide as the sulfhydryl group oxidant were used. Cyclic voltammetry measurements, following the redox state of glutathione, were performed on a hanging mercury drop electrode (HMDE) in borate buffer (pH 9.2). It was shown that in aqueous solutions TCEP was able to reduce disulfide groups smoothly and quantitatively. The TCEP response at -0.25 V vs. Ag/AgCl/3 M KCl did not disturb the signals of the thiol/disulfide redox couple. The origin of cathodic and anodic signals of GSH (at -0.44 and -0.37 V) and GSSG (at -0.69 and -0.40 V) glutathione forms is discussed. It was shown that the application of TCEP to the conservation of sulfhydryl groups in peptides and proteins can be useful instrument for the study of peptides and proteins redox behavior.     

Common prevalence of alanine and glycine in mobile reactive centre loops of serpins and viral fusion peptides: Do prions possess a fusion peptide?

Summary Serpin reactive centre loops and fusion peptides released by proteolytic cleavage are particularly mobile. Their amino acid compositions reveal a common and unusual abundance of alanine, accompanied by high levels of glycine. These two small residues, which are not simultaneously abundant in stable helices (standard or transmembrane), probably play an important role in mobility. Threonine and valine (also relatively small amino acids) are also abundant in these two kinds of peptides. Moreover, the known 3D structures of an uncleaved serpin reactive centre and a fusion peptide are strikingly similar. Such sequences possess many small residues and are found in several signal peptides and in PrP, a protein associated with spongi-form encephalopathies and resembling virus envelope proteins. These properties may be related to the infection mechanisms of these diseases.