A new detector for fully automatic peptide synthesis

A new method of monitoring the rate of reactions in solid-phase peptide synthesis is described. A conductivity detector in the reaction cell (patent applied for) enables the deprotection, washing and subsequent coupling stages to be examined in detail. The half-lives of the reactions can be calculated and hence the optimum reaction times can be predicted. Difficult sequences are sensed and appropriate action taken completely automatically.     

Synthesis of peptide aldehydes via enzymatic acylation of amino aldehyde derivatives

Two ways for semi-enzymatic preparation of the peptide aldehydes are proposed: (1) enzymatic acylation of amino alcohols with acyl peptide esters and subsequent chemical oxidation of the resulting peptide alcohols with DMSO/acetic anhydride mixture or (2) enzymatic acylation of the preliminarily obtained by a chemical route amino aldehyde semicarbazones. Subtilisin 72, serine proteinase with a broad specificity, distributed over macroporous silica, was used as a catalyst in both cases. Due to the practical absence of water in the reaction mixtures the yields of the products in both enzymatic reactions were nearly quantitative. The second way seems to be more attractive because all chemical stages were carried out with amino acid derivatives, far less valuable compounds than peptide ones. A series of peptide aldehydes of general formula Z-Ala-Ala-Xaa-al (where Xaa-al=leucinal, phenylalaninal, alaninal, valinal) was obtained. The inhibition parameters for these compounds, in the hydrolysis reactions of corresponding chromogenic substrates for subtilisin and -chymotrypsin, were determined.     

Peptide synthesis catalyzed by polyethylene glycol-modified chymotrypsin in organic solvents

Chymotrypsin modified with polyethylene glycol was successfully used for peptide synthesis in organic solvents. The benzene-soluble modified enzyme readily catalyzed both aminolysis of N-benzoyl-L-tyrosine p-nitroanilide and synthesis of N-benzoyl-L-tyrosine butylamide in the presence of trace amounts of water. A quantitative reaction was obtained when either hydrophobic or bulky amides of L- as well as D-amino acids were used as acceptor nucleophiles, while almost no reaction occurred with free amino acids or ester derivatives. The acceptor nucleophile specificity of modified chymotrypsin as a catalyst in the formation of both amide and peptide bonds in organic solvents was quite comparable to that in aqueous solution as well as to that of the leaving group in hydrolysis reactions. By contrast, the substrate specificity of modified chymotrypsin in organic solvents was different from that in water since arginine and lysine esters were found to be as effective as aromatic amino acids to form the acyl-enzyme with subsequent synthesis of a peptide bond.     

Synthesis of β-Peptide Standards for Use in Model Prebiotic Reactions

A one-pot method was developed for the preparation of a series of β-alanine standards of moderate size (2 to ≥12 residues) for studies concerning the prebiotic origins of peptides. The one-pot synthesis involved two sequential reactions: (1) dry-down self-condensation of β-alanine methyl ester, yielding β-alanine peptide methyl ester oligomers, and (2) subsequent hydrolysis of β-alanine peptide methyl ester oligomers, producing a series of β-alanine peptide standards. These standards were then spiked into a model prebiotic product mixture to confirm by HPLC the formation of β-alanine peptides under plausible reaction conditions. The simplicity of this approach suggests it can be used to prepare a variety of β-peptide standards for investigating differences between α- and β-peptides in the context of prebiotic chemistry.     

Limiting racemization and aspartimide formation in microwave-enhanced Fmoc solid phase peptide synthesis.

Microwave energy represents an efficient manner to accelerate both the deprotection and coupling reactions in 9-fluorenylmethyloxycarbonyl (Fmoc) solid phase peptide synthesis (SPPS). Typical SPPS side reactions including racemization and aspartimide formation can occur with microwave energy but can easily be controlled by routine use of optimized methods. Cysteine, histidine, and aspartic acid were susceptible to racemization during microwave SPPS of a model 20mer peptide containing all 20 natural amino acids. Lowering the microwave coupling temperature from 80 degrees C to 50 degrees C limited racemization of histidine and cysteine. Additionally, coupling of both histidine and cysteine can be performed conventionally while the rest of the peptide is synthesized using microwave without any deleterious effect, as racemization during the coupling reaction was limited to the activated ester state of the amino acids up to 80 degrees C. Use of the hindered amine, collidine, in the coupling reaction also minimized formation of D-cysteine. Aspartimide formation and subsequent racemization of aspartic acid was reduced by the addition of HOBt to the deprotection solution and/or use of piperazine in place of piperidine.     

Inhibition of glyceraldehyde-3-phosphate dehydrogenase by peptide and protein peroxides generated by singlet oxygen attack.

Reaction of certain peptides and proteins with singlet oxygen (generated by visible light in the presence of rose bengal dye) yields long-lived peptide and protein peroxides. Incubation of these peroxides with glyceraldehyde-3-phosphate dehydrogenase, in the absence of added metal ions, results in loss of enzymatic activity. Comparative studies with a range of peroxides have shown that this inhibition is concentration, peroxide, and time dependent, with H2O2 less efficient than some peptide peroxides. Enzyme inhibition correlates with loss of both the peroxide and enzyme thiol residues, with a stoichiometry of two thiols lost per peroxide consumed. Blocking the thiol residues prevents reaction with the peroxide. This stoichiometry, the lack of metal-ion dependence, and the absence of electron paramagnetic resonance (EPR)-detectable species, is consistent with a molecular (nonradical) reaction between the active-site thiol of the enzyme and the peroxide. A number of low-molecular-mass compounds including thiols and ascorbate, but not Trolox C, can prevent inhibition by removing the initial peroxide, or species derived from it. In contrast, glutathione reductase and lactate dehydrogenase are poorly inhibited by these peroxides in the absence of added Fe2+-EDTA. The presence of this metal-ion complex enhanced the inhibition observed with these enzymes consistent with the occurrence of radical-mediated reactions. Overall, these studies demonstrate that singlet oxygen-mediated damage to an initial target protein can result in selective subsequent damage to other proteins, as evidenced by loss of enzymatic activity, via the formation and subsequent reactions of protein peroxides. These reactions may be important in the development of cellular dysfunction as a result of photo-oxidation.     

Picornaviral processing: some new ideas

Mature picornaviral proteins are derived by progressive, post-translational cleavage of a giant precursor polyprotein. At least three viral-encoded proteolytic activities are involved in the processing. The first cleavage takes place while the polyprotein is still nascent on a ribosome. In poliovirus, this event is probably catalyzed by peptide 2A, a protein from the middle portion of the genome. Most subsequent processing is effected by viral protease 3C, a thiol-type enzyme, responsible for eight to ten self-cleaving and autocatalytic reactions within the polyprotein. The final proteolytic processing event, maturation of the VPO peptide, may occur by a novel, autocatalytic, serine-type mechanism, where viral RNA serves as proton-acceptor during the cleavage reaction.     

Kinetic analysis of beta-amyloid fibril elongation

We used surface plasmon resonance biosensors to evaluate the kinetics associated with the initial events of beta-amyloid (Abeta) fibril elongation. Fibrils were immobilized on the sensor chip surface and extended by exposure to soluble Abeta(1-40) peptide. The fibril surfaces bound Congo red, a marker for beta sheet structures, and exhibited a slow linear background decay that is consistent with fibril depolymerization. Sonicated fibrils supported elongation better than unsonicated fibrils, which is consistent with fibril extension reactions. The kinetic data revealed that peptide association and dissociation occurred in multiple steps. Kinetic rate constants for fibril extension were determined by globally fitting the response data with a three-step polymerization model. In the first step, the soluble peptide binds to the growing fibril tip in a readily reversible reaction. The subsequent steps likely allow bound peptide to be stabilized into the growing fiber through postbinding transitional events. Using a mutant peptide, F19P Abeta(1-40), we illustrate how the biosensor assay can be used to probe structure/function relationships of fibril elongation.     

Spectrophotometric determination of tryptophan and tyrosine in peptides and proteins based on new color reactions

A new spectrophotometric method for quantitative determination of tryptophan and tyrosine in peptides and proteins is described. It is based on two specific color reactions, the reaction of tryptophan with formaldehyde and the reaction of tyrosine and tryptophan with hydroxylamine and ceric cations. By combination of these two reactions both tyrosine and tryptophan can be determined simultaneously. Tyrosine and/or tryptophan bound in peptides and/or proteins react independently of the rest of the peptide or protein molecule. The method is simple, accurate, and sensitive. Hydrolysis is not necessary.     

Selective determination of arginine-containing and tyrosine-containing peptides using capillary electrophoresis and laser-induced fluorescence detection.

The use of two different amino acid-selective fluorogenic reagents for the derivatization of peptides is investigated. One such scheme utilizes a selective reaction of benzoin with the guanidine moiety to derivatize arginine residues occurring in a peptide. The second scheme involves the formylation of tyrosine, followed by reaction with 4-methoxy-1,2-phenylenediamine. The use of capillary electrophoresis and laser-induced fluorescence detection allows enhanced efficiencies and sensitivities to be obtained for the separations of either arginine- or tyrosine-containing peptides. A helium-cadmium laser (325 nm) is ideally suited for the laser-based detection system due to a close match of the excitation maxima of derivatized peptides from both reactions. A detection limit of 270 amol is achieved for model arginine-containing peptides, while the detection limit for model tyrosine-containing peptides is measured at 390 amol. Both derivatization reactions are found to be useful for high-sensitivity peptide mapping applications in which only the peptides containing the derivatized amino acids are detected.     

Methionine ligation strategy in the biomimetic synthesis of parathyroid hormones

In biological systems, both proteolysis and aminolysis of amide bonds produce activated intermediates through acyl transfer reactions either inter- or intramolecularly. Protein splicing is an illustrative example that proceeds through a series of catalyzed acyl transfer reactions and culminates at an O- or S-acyl intermediate. This intermediate leads to an uncatalyzed acyl migration to form an amide bond in the spliced product. A ligation method mimicking the uncatalyzed final steps in protein splicing has been developed utilizing the acyl transfer amide-bond feature for the blockwise coupling of unprotected, free peptide segments at methionine (Met). The latent thiol moiety of Met can be exploited using homocysteine at the α-amino terminal position of a free peptide for transthioesterification with another free peptide containing an α-thioester to give an S-acyl intermediate. A subsequent, proximity-driven S- to N-acyl migration of this acyl intermediate spontaneously rearranges to form a homocysteinyl amide bond. S-methylation with excess p-nitrobenezensulfonate yields Met at the ligation site. The methionine ligation is selective and orthogonal, and is usually completed within 4 h when performed at slightly basic pH and under strongly reductive conditions. No side reactions due to acylation were observed with any other α-amines of both peptide segments as seen in the synthesis of parathyroid hormone peptides. Furthermore, cyclic peptide can also be obtained through the same strategy by placing both homocysteine at the amino terminus and the thioester at the carboxyl terminus in an unprotected peptide precursor. These biomimetic ligation strategies hold promise for engineering novel peptides and proteins. © 1998 John Wiley & Sons, Inc. Biopoly 46: 319–327, 1998     

Generation of peptide-MHC class I complexes through UV-mediated ligand exchange

Major histocompatibility complex (MHC) class I molecules present peptide ligands on the cell surface for recognition by appropriate cytotoxic T cells. MHC-bound peptides are critical for the stability of the MHC complex, and standard strategies for the production of recombinant MHC complexes are based on in vitro refolding reactions with specific peptides. This strategy is not amenable to high-throughput production of vast collections of MHC molecules. We have developed conditional MHC ligands that form stable complexes with MHC molecules but can be cleaved upon UV irradiation. The resulting empty, peptide-receptive MHC molecules can be charged with epitopes of choice under native conditions. Here we describe in-depth procedures for the high-throughput production of peptide-MHC (pMHC) complexes by MHC exchange, the analysis of peptide exchange efficiency by ELISA and the parallel production of MHC tetramers for T-cell detection. The production of the conditional pMHC complex by an in vitro refolding reaction can be achieved within 2 weeks, and the actual high-throughput MHC peptide exchange and subsequent MHC tetramer formation require less than a day.     

N-Terminal Protein Characterization by Mass Spectrometry after Cyanogen Bromide Cleavage using Combined Microscale Liquid- and Solid-Phase Derivatization

A sample preparation method for protein N-terminal peptide isolation from cyanogen bromide (CNBr) protein digests has been developed. In this strategy, the CNBr cleavage was preceded by protein α- and ε-amine acetylation and carboxyamidomethylation in a one-pot reaction scheme. The peptide mixture was adsorbed on ZipTip_C18 pipette tips for reaction of the newly generated N-termini with sulfosuccinimidyl-2-(biotinamido) ethyl-1, 3-dithiopropionate. In the subsequent steps, the peptides were exposed in situ to hydroxylamine for reversal of potential hydroxyl group acylation, followed by reductive release of the disulfide-linked biotinamido moiety from the derivatives. The selectively thiol group-functionalized internal and C-terminal peptides were reversibly captured by covalent chromatography on activated thiol-sepharose, leaving the N-terminal fragment in the flow-through fraction. The use of the reversed-phase support as a venue for postcleavage serial modification proved instrumental to ensure throughput and completeness of derivatization. By this sequence of solid-phase reactions, the N-terminal peptide could be recognized uniquely in the MALDI-mass spectra of unfractionated digests by its unaltered mass signature. The use of the sample preparation method was demonstrated with low-picomole amounts of model protein. The N-terminal CNBr fragments were retrieved selectively from the affinity support. The sample preparation method provides for robustness and simplicity of operation using standard equipment available in most biological laboratories and is anticipated to be readily expanded to gel-separated proteins.     

Kinetics of the inhibition of plasminogen activators by the plasminogen-activator inhibitor. Evidence for ''second-site'' interactions.

The reactions between plasminogen-activator inhibitor (PAI) and different plasminogen activators were studied in the presence of chromogenic peptide substrates for the enzymes. Our findings suggest that the rate constants for the reactions of PAI with single-chain tissue plasminogen activator (tPA), two-chain tPA, high-Mr urokinase and low-Mr urokinase are high and quite similar (1.6 X 10(7)-3.9 X 10(7) M-1.s-1). A free active site in the enzymes seems to be necessary for their reaction with PAI. Amino acids with antifibrinolytic properties did not interfere with the reactions. However, di-isopropyl phosphorofluoridate-inactivated tPA inhibited the reaction between PAI and all plasminogen activators in a similar way. These findings clearly demonstrated that a 'second-site' interaction, in addition to that between the enzyme active site and the inhibitor 'bait' peptide bond, is of importance for the high reaction rate. The reaction rate between PAI and single-chain tPA in the presence of an activator substrate (D-Ile-Pro-Arg p-nitroanilide) was decreased in the presence of fibrin. Fibrin caused a decrease in the Km for the single-chain tPA-substrate reaction. As a consequence, the 'free' concentration of single-chain tPA in the system decreased in the presence of fibrin, affecting the reaction rate between PAI and single-chain tPA. The phenomenon might be of physiological relevance, in the sense that single-chain tPA bound to fibrin in the presence of plasminogen would be protected against inactivation by PAI.     

Kinetic Analysis of Self-Replicating Peptides: Possibility of Chiral Amplification in Open Systems

A simplified kinetic model scheme is presented that addresses the main reactions of two recently reported peptide self-replicators. Experimentally observed differences in the autocatalytic efficiency between these two systems-- caused by variations in the peptide sequences--and the possible effect of chiral amplification under heterochiral reaction conditions were evaluated. Our numerical simulations indicated that differences in the catalytic performance are exclusively due to pronounced variations in the rate parameters that control the reversible and hydrophobic interactions in the reaction system but neither to alterations in the underlying reaction network nor to changes in the stoichiometry of the involved aggregation processes. Model predictions further demonstrated the possible existence of chiral amplification if peptide self-replication is performed under heterochiral reaction conditions. Pointing into the direction of a possible cause for biomolecular homochirality, it was found that in open flow reactors, keeping the system under non-equilibrium conditions, a remarkable amplification of enantiomeric excess could be achieved. According to our modeling, this is due to a chiroselective autocatalytic effect and a meso-type separation process both of which are assumed to be intrinsic for the underlying dynamics of heterochiral peptide self-replication.     

Autocatalytic sets of proteins

This article investigates the possibility that the emergence of reflexively autocatalytic sets of peptides and polypeptides may be an essentially inevitable collective property of any sufficiently complex set of polypeptides. The central idea is based on the connectivity properties of random directed graphs. In the set of amino acid monomer and polymer species up to some maximum length, M, the number of possible polypeptides is large, but, for specifiable "legitimate" end condensation, cleavage and transpeptidation exchange reactions, the number of potential reactions by which the possible polypeptides can interconvert is very much larger. A directed graph in which arrows from smaller fragments to larger condensation products depict potential synthesis reactions, while arrows from the larger peptide to the smaller fragments depict the reverse cleavage reactions, comprises the reaction graph for such a system. Polypeptide protoenzymes are able to catalyze such reactions. The distribution of catalytic capacities in peptide space is a fundamental problem in its own right, and in its bearing on the existence of autocatalytic sets of proteins. Using an initial idealized hypothesis that an arbitrary polypeptide has a fixed a priori probability of catalyzing any arbitrary legitimate reaction to assign to each polypeptide those reactions, if any, which it catalyzes, the probability that the set of polypeptides up to length M contains a reflexively autocatalytic subset can be calculated and is a percolation problem on such reaction graphs. Because, as M increases, the ratio of reactions among the possible polypeptides to polypeptides rises rapidly, the existence of such autocatalytic subsets is assured for any fixed probability of catalysis. The main conclusions of this analysis appear independent of the idealizations of the initial model, introduce a novel kind of parallel selection for peptides catalyzing connected sequences of reactions, depend upon a new kind of minimal critical complexity whose properties are definable, and suggest that the emergence of self replicating systems may be a self organizing collective property of critically complex protein systems in prebiotic evolution. Similar principles may apply to the emergence of a primitive connected metabolism. Recombinant DNA procedures, cloning random DNA coding sequences into expression vectors, afford a direct avenue to test the distribution of catalytic capacities in peptide space, may provide a new means to select or screen for peptides with useful properties, and may ultimately lead toward the actual construction of autocatalytic peptide sets.     

Chemical modification of the haem propionate of cytochrome c. A re-evaluation of the reaction of cytochrome c with a water-soluble carbodi-imide.

Horse heart and tuna heart cytochromes c were treated with the water-soluble carbodi-imide 1-(3-dimethylaminopropyl)-3-ethylcarbodi-imide. When the reaction is followed spectroscopically two kinetic phases are apparent. Alteration of the reactivity of the proteins with such ligands as CO, however, occurs in a single phase identical with the faster phase detected spectroscopically. The modified proteins both show spectroscopic and redox properties identical with those described for the tuna heart cytochrome c derivative by Timkovich [Biochem. J. (1980) 185, 47-57]. The use of radiolabelled carbodi-imide identifies two or three sites of reactivity. However, the addition of glycine methyl ester to the reaction mixture leads to the addition of nine glycine moieties in the case of the horse protein and seven in the case of the tuna protein, indicating a larger number of reactive sites than previously reported. A further set of reaction sites was identified by peptide mapping of the modified proteins, and these sites take part in intramolecular reactions leading to internal cross-linking and the formation of an enzymically indigestible 'core particle'. The haem group was identified as a site of reaction with the carbodi-imide, and is as a consequence covalently linked to the peptide by a bond in addition to the thioether bonds normally present. In the light of these findings, the alterations in the properties of the tuna protein, subsequent to reaction with the carbodi-imide, which have been previously explained in structural terms, must be re-evaluated. This study also highlights the importance of internal cross-link formation, which can occur by intramolecular nucleophilic attack, a process that has often been overlooked by investigators employing carbodi-imide modification of carboxylate groups in proteins.     

C-Terminal Protein Characterization by Mass Spectrometry: Isolation of C-Terminal Fragments from Cyanogen Bromide-Cleaved Protein

A sample preparation method for protein C-terminal peptide isolation from cyanogen bromide (CNBr) digests has been developed. In this strategy, the analyte was reduced and carboxyamidomethylated, followed by CNBr cleavage in a one-pot reaction scheme. The digest was then adsorbed on ZipTip_C18 pipette tips for conjugation of the homoserine lactone-terminated peptides with 2,2′-dithiobis (ethylamine) dihydrochloride, followed by reductive release of 2-aminoethanethiol from the derivatives. The thiol-functionalized internal and N-terminal peptides were scavenged on activated thiol sepharose, leaving the C-terminal peptide in the flow-through fraction. The use of reversed-phase supports as a venue for peptide derivatization enabled facile optimization of the individual reaction steps for throughput and completeness of reaction. Reagents were replaced directly on the support, allowing the reactions to proceed at minimal sample loss. By this sequence of solid-phase reactions, the C-terminal peptide could be recognized uniquely in mass spectra of unfractionated digests by its unaltered mass signature. The use of the sample preparation method was demonstrated with low-level amounts of a whole, intact model protein. The C-terminal fragments were retrieved selectively and efficiently from the affinity support. The use of covalent chromatography for C-terminal peptide purification enabled recovery of the depleted material for further chemical and/or enzymatic manipulation. The sample preparation method provides for robustness and simplicity of operation and is anticipated to be expanded to gel-separated proteins and in a scaled-up format to high-throughput protein profiling in complex biological mixtures.     

Aqueous Synthesis of Peptide Thioesters from Amino Acids and a Thiol Using 1,1′-Carbonyldiimidazole

A new method was developed for the synthesis of peptide thioesters from free amino acids and thiols in water. This one-pot simple method involves two steps: (1) activation in water of an amino acid presumably as its N-carboxyanhydride (NCA) using 1,1′-carbonyldiimidazole (CDI), and (2) subsequent condensation of the activated amino acid-NCA in the presence of a thiol. With this method citrulline peptide thioesters containing up to 10 amino acid residues were prepared in a single reaction. This aqueous synthetic method provides a simple way to prepare peptide thioesters for studies of peptide replication involving ligation of peptide thioesters on peptide templates. The relevance of peptide replication to the origin-of-life process is supported by previous studies showing that amino acid thioesters (peptide thioester precursors) can be synthesized under prebiotic conditions by reaction of small sugars with ammonia and a thiol.     

The metal-catalyzed oxidation of methionine in peptides by Fenton systems involves two consecutive one-electron oxidation processes.

The one-electron oxidation of methionine (Met) plays an important role in the redox reactions of Met in peptides and proteins under conditions of oxidative stress, e.g., during the metal-catalyzed oxidation of beta-amyloid peptide (beta A). However, little information is available with regard to mechanisms and product formation during the metal-catalyzed oxidation of Met. Here, we demonstrate that two-electron oxidation of Met in Fenton reactions, carried out aerobically by [Fe(II)(EDTA)](2-) and H(2)O(2) (EDTA = ethylenediaminetetra acetate) is the consequence of two consecutive one-electron transfer reactions carried out by either free or complexed hydroxyl radicals, followed by the reaction of an intermediary sulfur-nitrogen bonded radical cation (sulfuranyl radical) with O(2). The model peptide Met-Met represents an ideal substrate for these investigations as its one-electron oxidation, followed by reaction with molecular oxygen, produces unique intermediates, azasulfonium diastereomers, which can be chemically isolated before hydrolysis to sulfoxide occurs.