Effect of freezing on the enzymatic coupling of specific amino acid-containing peptide fragments

The effect of freezing on the enzymatic coupling of highly specific amino acid-containing peptide fragments was investigated using trypsin, α-chymotrypsin, and Bacillus licheniformis Glu-specific endopeptidase as biocatalysts. Comparison with reactions at normal temperature indicates that freezing efficiently represses the cleavage of specific peptide bonds independent of their individual localisation and specificity achieving irreversible and efficient peptide bond formation without proteolytic side reactions.     

Substrate mimetics in protease catalysis: characteristics,kinetics, and synthetic utility

This article reviews the latest developments in protease-catalyzed peptide synthesis focusing on the use of substrate mimetics. The substrate mimetics approach takes advantage of the characteristic of this novel type of substrates to direct the enzyme to recognize an alternative site on the acyl donor, i.e. the site-specific ester leaving group, mediating the acceptance of originally poorly reactive acyl moieties. At first the kinetics and catalytic mechanism of substrate mimetics-mediated reactions are discussed on the basis of hydrolysis, peptide synthesis, protein-ligand docking, and molecular dynamics studies. By the example of the Glu-specific V8 protease and the aromatic amino acid-specific chymotrypsin both the empirical and computer-aided design of specific substrate mimetics is described. The influence of the leaving group specifically recognized by the enzyme is also considered. The benefits of these artificial substrates over common acyl donor components are illustrated by selected synthesis reactions of small peptides, peptide isosteres, non-peptidic carboxylic acid amides, and the coupling of peptide fragments at non-specific ligation sites resulting in biologically active peptide products. Finally, this review focuses on critical syntheses that uses specific-amino acid-containing peptides as the reactants of ligation. Based on these, the restrictions of the substrate mimetics approach is critically discussed and techniques to their overcoming are presented.     

Reverse proteolysis promoted by in situ generated peptide ester fragments

In this contribution we describe a general synthesis concept for the in situ preparation of protease specific reactants using methyl thioesters as universal precursors. The precursor esters are readily available by standard synthesis procedures and can be used directly as reactants for protease-mediated peptide coupling reactions. Alternatively, they can serve as initial building blocks for the in situ preparation of various types of substrate mimetics. The synthesis of the latter is achieved by a one-pot spontaneous transthioesterification reaction of the parent thioester (Y-(Xaa)(n)-SMe-->Y-(Xaa)(n)-SR; R: CH(2)CH(2)COOH, CH(2)C(6)H(5), C(6)H(4)NHC(:NH)NH(2)), which proceeds efficiently in both a sequential manner and parallel to the subsequent enzymatic reaction. The resulting substrate mimetics act as efficient acyl donor components and show the typical behavior of substrate mimicry enabling irreversible reactions with originally nonspecific acyl moieties. Neither a workup of the substrate mimetic intermediate nor changes of the reaction conditions during the whole synthesis process are required. Model peptide syntheses using trypsin, alpha-chymotrypsin, and V8 protease as the biocatalysts proved the function of the approach and illustrated its synthetic value for protease-mediated reactions and the compatibility of the approach with state-of-the-art solid-phase peptide ester synthesis methods.     

Sequence dependent fragmentation of peptides generated by MALDI quadrupole time-of-flight (MALDI Q-TOF) mass spectrometry and its implications for protein identification

A study has been undertaken to evaluate the usefulness of MALDI Q-TOF data for protein identification. The comparison of MS data of protein digests obtained on a conventional MALDI TOF instrument to the MS data from the MALDI Q-TOF reveal peptide patterns with similar intensity ratios. However, comparison of MS/MS Q-TOF data produced by nanoelectrospray versus MALDI reveals striking differences. Peptide fragment ions obtained from doubly charged precursors produced by nanoelectrospray are mainly y-type ions with some b-ions in the lower mass range. In contrast, peptide fragment ions produced from the singly charged ions originating from the MALDI source are a mixture of y-, b- and a-ions accompanied by ions resulting from neutral loss of ammonia or water. The ratio and intensity of these fragment ions is found to be strongly sequence dependent for MALDI generated ions. The singly charged peptides generated by MALDI show a preferential cleavage of the C-terminal bond of acidic residues aspartic and glutamic acid and the N-terminal bond of proline. This preferential cleavage can be explained by the mobile proton model and is present in peptides that contain both arginine and an acidic amino acid. The MALDI Q-TOF MS/MS data of 24 out of 26 proteolytic peptides produced by trypsin or Asp-N digestions were successfully used for protein identification via database searching, thus indicating the general usefulness of the data for protein identification. De novo sequencing using a mixture of 160/18O water during digestion has been explored and de novo sequences for a number of peptides have been obtained.     

Sequencing Lys-N Proteolytic Peptides by ESI and MALDI Tandem Mass Spectrometry

In this study, we explored the MS/MS behavior of various synthetic peptides that possess a lysine residue at the N-terminal position. These peptides were designed to mimic peptides produced upon proteolysis by the Lys-N enzyme, a metalloendopeptidase issued from a Japanese fungus Grifola frondosa that was recently investigated in proteomic studies as an alternative to trypsin digestion, as a specific cleavage at the amide X-Lys chain is obtained that provides N-terminal lysine peptide fragments. In contrast to tryptic peptides exhibiting a lysine or arginine residue solely at the C-terminal position, and are thus devoid of such basic amino acids within the sequence, these Lys-N proteolytic peptides can contain the highly basic arginine residue anywhere within the peptide chain. The fragmentation patterns of such sequences with the ESI-QqTOF and MALDI-TOF/TOF mass spectrometers commonly used in proteomic bottom-up experiments were investigated.     

Protease-catalyzed peptide synthesis for the site-specific incorporation of alpha-fluoroalkyl amino acids into peptides

Substitution of native amino acids by fluoroalkyl analogues represents a new approach for the design of biologically active peptides with increased metabolic stability as well as defined secondary structure and provides a powerful label for spectroscopic investigations. Here, we introduce a methodology for the incorporation of sterically demanding C(alpha)-fluoroalkyl amino acids into the P(1) position of peptides catalyzed by the commercially available proteases trypsin and alpha-chymotrypsin. The combination of 4-guanidinophenyl ester of C(alpha)-fluoroalkyl amino acids as substrate mimetics with frozen-state reaction conditions provided the most efficient strategy for protease-catalyzed site-specific introduction of this kind of nonnatural amino acids into peptide sequences. Consequently, a library of di-, tri-, and tetrapeptides containing alpha-methyl, alpha-difluoromethyl, and alpha-trifluoromethyl alanine, leucine, and phenylalanine in the P(1) position was synthesized catalyzed by trypsin as well as alpha-chymotrypsin. Trypsin was shown to be the more versatile protease.     

Energetic switch of the proline effect in collision‐induced dissociation of singly and doubly protonated peptide Ala‐Ala‐Arg‐Pro‐Ala‐Ala

Suppression of the selective cleavage at N‐terminal of proline is observed in the peptide cleavage by proteolytic enzyme trypsin and in the fragment ion mass spectra of peptides containing Arg‐Pro sequence. An insight into the fragmentation mechanism of the influence of arginine residue on the proline effect can help in prediction of mass spectra and in protein structure analysis. In this work, collision‐induced dissociation spectra of singly and doubly charged peptide AARPAA were studied by ESI MS/MS and theoretical calculation methods. The proline effect was evaluated by comparing the experimental ratio of fragments originated from cleavage of different amide bonds. The results revealed that the backbone amide bond cleavage was selected by the energy barrier height of the fragmentation pathway although the strong proton affinity of the Arg side chain affected the stereostructure of the peptide and the dissociation mechanism. The thermodynamic stability of the fragment ions played a secondary role in the abundance ratio of fragments generated via different pathways. Fragmentation studies of protonated peptide AACitPAA supported the energy‐dependent hypothesis. The results provide an explanation to the long‐term arguments between the steric conflict and the proton mobility mechanisms of proline effect.     

Expanded structural and stereospecificity in peptide synthesis with chemically modified mutants of subtilisin

Employing the strategy of combined site directed mutagenesis and chemical modification, we previously generated chemically modified mutant enzymes (CMMs) of subtilisin Bacillus lentus (SBL). We now report the use of these SBL-CMMs for peptide coupling reactions. The SBL-CMMs exhibit dramatically altered substrate specificity, including the acceptance of d-amino acid acyl donors, generating dipeptides containing d-Phe, d-Ala and d-Glu in up to 66% yield, which was not possible using wild-type SBL (WT-SBL). In addition, SBL-CMMs accommodate α-branched amino acids such as l-Ala-NH₂ as acyl acceptors in their S₁′ pockets, which WT-SBL will not.     

Effects of disulfide linkages on gas-phase reactions of small multiply charged peptide ions

Multiply protonated ions of disulfide-intact and -reduced peptides were generated by electrospray ionization and studied by Fourier transform ion cyclotron resonance mass spectrometry. The effects of disulfide bonds on gas-phase deprotonation reactions and hydrogen/deuterium (H/D) exchange were investigated. Insight into conformations was gained from molecular dynamics calculations. For ions from three small peptides containing 9–14 amino acid residues, H/D exchange is more sensitive to changes in conformation than deprotonation. However, with both gas-phase reactions the more diffuse forms of the peptides (as determined by molecular modeling) react more readily. The effects of disulfide cleavage on the conformations and on the reactions were found to depend upon the sequence of the peptide. For [M + 3H]³⁺ of TGF-α (34–43), reduction of the disulfide linkage leads to a greatly extended structure and a dramatic increase in the rate and extent of H/D exchange. In contrast, [M + 2H]²⁺ of Arg⁸ -vasopressin becomes slightly more compact upon cleavage of the disulfide bond; these reduced ions are slower to react. For [M + 3H]³⁺ of somatostatin-14, reduction of the disulfide bond has little effect on conformation or gas-phase reactivity. Overall, these results indicate that there is no general rule on how cleavage of a disulfide bond will effect a peptide ion’s gas-phase reactivity.     

Evaluation of the influence of amino acid composition on the propensity for collision-induced dissociation of model peptides using molecular dynamics simulations

The dynamical behavior of model peptides was evaluated with respect to their ability to form internal proton donor-acceptor pairs using molecular dynamics simulations. The proton donor-acceptor pairs are postulated to be prerequisites for peptide bond cleavage resulting in formation of b and y ions during low-energy collision-induced dissociation in tandem mass spectrometry (MS/MS). The simulations for the polyalanine pentamer Ala(5)H(+) were compared with experimental data from energy-resolved surface induced dissociation (SID) studies. The results of the simulation are insightful into the events that likely lead up to the fragmentation of peptides. Nine-mer polyalanine-based model peptides were used to examine the dynamical effect of each of the 20 common amino acids on the probability to form donor-acceptor pairs at labile peptide bonds. A range of probabilities was observed as a function of the substituted amino acid. However, the location of the peptide bond involved in the donor-acceptor pair plays a critical role in the dynamical behavior. This influence of position on the probability of forming a donor-acceptor pair would be hard to predict from statistical analyses on experimental spectra of aggregate, diverse peptides. In addition, the inclusion of basic side chains in the model peptides alters the probability of forming donor-acceptor pairs across the entire backbone. In this case, there are still more ionizing protons than basic residues, but the side chains of the basic amino acids form stable hydrogen bond networks with the peptide carbonyl oxygens and thus act to prevent free access of "mobile protons" to labile peptide bonds. It is clear from the work that the identification of peptides from low-energy CID using automated computational methods should consider the location of the fragmenting bond as well as the amino acid composition.     

Peptide bond formation by aminolysin-A catalysis: a simple approach to enzymatic synthesis of diverse short oligopeptides and biologically active puromycins

A new S9 family aminopeptidase derived from the actinobacterial thermophile Acidothermus cellulolyticus was cloned and engineered into a transaminopeptidase by site-directed mutagenesis of catalytic Ser(491) into Cys. The engineered biocatalyst, designated aminolysin-A, can catalyze the formation of peptide bonds to give linear homo-oligopeptides, hetero-dipeptides, and cyclic dipeptides using cost-effective substrates in a one-pot reaction. Aminolysin-A can recognize several C-terminal-modified amino acids, including the l- and d-forms, as acyl donors as well as free amines, including amino acids and puromycin aminonucleoside, as acyl acceptors. The absence of amino acid esters prevents the formation of peptides; therefore, the reaction mechanism involves aminolysis and not a reverse reaction of hydrolysis. The aminolysin system will be a beneficial tool for the preparation of structurally diverse peptide mimetics by a simple approach.     

Chemical ionization mass spectrometry of complex molecules—VI: Peptides

The chemical ionization mass spectra of a series of simple peptides containing six or fewer amino acids have been studied. Using methane as the reactant gas we found cleavage of the peptide bond occurs in two ways, yielding either the acyl carbonium ion or the complementary ammonium ion. The observation of both types of fragments permits the determination of the amino acid sequence of the peptide. The ammonium ions provide an additional sequence determining route compared to that available from electron-impact spectra. ‘Sequence-determing ions,’ especially the quasimolecular ion at m/e [M+1] are usually more intense than in the electron-impact mass spectra.     

Solid-phase synthesis of peptide and glycopeptide thioesters through side-chain-anchoring strategies

An efficient new strategy for the synthesis of peptide and glycopeptide thioesters is described. The method relies on the side-chain immobilization of a variety of Fmoc-amino acids, protected at their C-termini, on solid supports. Once anchored, peptides were constructed using solid-phase peptide synthesis according to the Fmoc protocol. After unmasking the C-terminal carboxylate, either thiols or amino acid thioesters were coupled to afford, after cleavage, peptide and glycopeptide thioesters in high yields. Using this method a significant proportion of the proteinogenic amino acids could be incorporated as C-terminal amino acid residues, therefore providing access to a large number of potential targets that can serve as acyl donors in subsequent ligation reactions. The utility of this methodology was exemplified in the synthesis of a 28 amino acid glycopeptide thioester, which was further elaborated to an N-terminal fragment of the glycoprotein erythropoietin (EPO) by native chemical ligation.     

Stereoselective synthesis of (Z)-alkene-containing proline dipeptide mimetics

In peptides and proteins, the peptide bond between an amino acid and proline exists as an equilibrium mixture of the cis-imide and trans-imide due to the low energy barrier in their interconversion. This feature greatly influences the structure and function of the proline-containing peptides and proteins. Therefore, restricting the amide bond with an (E)- or (Z)-alkene should provide a promising method for elucidating the structure-activity relationships of the peptide and the proteins. In this report, the regio- and stereoselective synthesis of cis-alanylproline (Ala-Pro) type (Z)-alkene dipeptide mimetic is described. The key steps of this synthesis are to introduce a C3 unit onto a gamma-phosphoryloxy-alpha,beta-unsaturated-delta-lactam with an organocopper-mediated anti-S(N)2' reaction and subsequently construct a five-membered proline-like cyclic structure with an intramolecular Suzuki coupling reaction. Hydrolysis of the amide bond in the resulting bicyclic lactam yields the desired cis-Ala-Pro type (Z)-alkene dipeptide isostere. The presented synthetic methodology should be applicable to the general syntheses of other cis-aminoacylproline type (Z)-alkene dipeptide mimetics.     

Substrate mimetics and freezing strategy: a useful combination that broadens the scope of proteases for synthesis

[reaction: see text] We present an irreversible and efficient protease-based method for peptide synthesis which occurs independently of the primary specificity of proteases and also without proteolytic side reactions. The key feature of this approach is the combination of the substrate mimetics strategy with frozen state enzymology. Model reactions catalyzed by several proteases qualify this approach as a powerful concept in the direction of a more universal application of proteases as biocatalysts for peptide ligation.     

Catalytic C−H/C−F Coupling of Azoles and Acyl Fluorides

A method for the palladium/copper-catalyzed direct acylation of azoles with acyl fluorides is described. This study reports the first examples of acyl fluorides being used as acylation reagents in transition-metal-catalyzed aromatic C−H bond functionalization reactions. Depending on the reaction temperature, decarbonylative coupling may also occur. Mechanistic studies suggest that the cleavage of the aromatic C−H bond, promoted by a copper-phosphine species, is not the rate-limiting step of this acylation.     

Acyl Fluorides in Late‐Transition‐Metal Catalysis

In this Review, we summarize the current state of the art in late‐transition‐metal‐catalyzed reactions of acyl fluorides, covering both their synthesis and further transformations. In organic reactions, the relationship between stability and reactivity of the starting substrates is usually characterized by a trade‐off. Yet, acyl fluorides display a very good balance between these properties, which is mostly due to their moderate electrophilicity. Thus, acyl fluorides (RCOF) can be used as versatile building blocks in transition‐metal‐catalyzed reactions, for example, as an “RCO” source in acyl coupling reactions, as an “R” source in decarbonylative coupling reactions, and as an “F” source in fluorination reactions. Starting from the cleavage of the acyl C?F bond in acyl fluorides, various transformations are accessible, including C?C, C?H, C?B, and C?F bond‐forming reactions that are catalyzed by transition‐metal catalysts that contain the Group 9–11 metals Co, Rh, Ir, Ni, Pd, or Cu.     

Trypsin-catalyzed synthesis of dipeptide containing alpha-aminoisobutyric acid using p- and m-(amidinomethyl)phenyl esters as acyl donor

Two series of inverse substrates, p- and m-(amidinomethyl)phenyl esters derived from N-(tert-butyloxycarbonyl)amino acid, were prepared as acyl donor components for enzymatic peptide synthesis. They were found to be readily coupled with an acyl acceptor such as L-alanine p-nitroanilide to produce dipeptide. An alpha-aminoisobutyric acid containing dipeptide was especially obtained in satisfactory yield. Streptomyces griseus trypsin was a more efficient catalyst than the bovine trypsin. The optimum condition for the coupling reaction was studied by changing the organic solvent, pH, and acyl acceptor concentration. It was found that the enzymatic hydrolysis of the resulting product was negligible.     

Peptidsynthesen mit immobilisierten Enzymen II. Immobilisiertes Trypsin,Thermolysin und Papain

Model studies were performed on the utility of covalently immobilized trypsin, thermolysin and papain for peptide bond formation. Trypsin and thermolysin catalyzed the formation of peptide bonds with nearly the same efficiency as the soluble proteases and they could be re-used successfully for further coupling experiments. The possibility of using immobilized trypsin and papain for kinetically controlled peptide bond formation was investigated. With the serine type enzyme trypsin excellent product yields were obtained starting with ester carboxyl components and an economical ratio of substrates. Experiments with the thiol protease papain were unsatisfactory because the once formed product is hydrolyzed as fast as the starting ester substrate used.     

Transition-metal complexes as enzyme-like reagents for protein cleavage: complex cis-[Pt(en)(H2O)2]2+ as a new methionine-specific protease

Complex cis-[Pt(en)(H(2)O)(2)](2+) promotes selective hydrolytic cleavage of two proteins, horse cytochrome c and bovine beta-casein. The cleavage is completed in 24 h under relatively mild conditions, at about pH 2.5, and a temperature as low as 40 degrees C. The results of HPLC and TSDS PAGE separations, MALDI mass spectrometry, and Edman sequencing showed that cleavage occurred exclusively at the peptide bond involving the C-terminus of each methionine residue, both such residues in cytochrome c and all six such residues in beta-casein. While having the same selectivity as cyanogen bromide (CNBr), the most common chemical protease, cis-[Pt(en)(H(2)O)(2)](2+) has several advantages. It is nonvolatile, easy to handle, and recyclable. Its cleavage is residue-selective, the rest of the polypeptide backbone remains intact, and the other side chains remain unmodified. It is applied in approximately equimolar amounts with respect to methionine residues, creates free amino and carboxylic groups, and cleaves even the Met-Pro bond, which is resistant to CNBr and most proteolytic enzymes. Finally the complex also works in the presence of the denaturing reagent sodium dodecyl sulfate. Experiments with the synthetic peptides, AcAla-Lys-Tyr-Gly-Gly-Met-Ala-Ala-Arg-Ala (termed Met-peptide) and AcVal-Lys-Gly-Gly-His-Ala-Lys-Tyr-Gly-Gly-Met-Ala-Ala-Arg-Ala (termed HisMet-peptide) as substrates, revealed structural and mechanistic features of the proteolytic reactions. We explain why two similar complexes with similar metal ions, cis-[Pt(en)(H(2)O)(2)](2+) and cis-[Pd(en)(H(2)O)(2)](2+), differ in selectivity as proteolytic reagents. The selectivity of cleavage is governed by the selectivity of the cis-[Pt(en)(H(2)O)(2)](2+) binding to the methionine side chain. The proteolytic activity is governed by the modes of coordination, which control the approach of the anchored Pt(II) ion to the scissile peptide bond. The cleavage occurs with a small, but significant, catalytic turnover of more than 18 after 7 days. The ability of cis-[Pt(en)(H(2)O)(2)](2+) to cleave proteins at relatively few sites, with explicable selectivity and catalytic turnover, bodes well for its use in biochemical practice.