Synthesis of cyclopentapeptides and cycloheptapeptides by DEPBT and the influence of some factors on cyclization.

Three cyclic peptides - cyclo(GlyAlaTyrLeuAla), cyclo(GlyProTyrLeuAla) and cyclo(GlyTyrGlyGlyProPhePro) - isolated and identified from medicinal herbs were chosen as model cyclic peptides to study the influence of the linear precursors and coupling reagents on cyclization. The 17 linear precursors of these three cyclic peptides were synthesized and cyclized using 3-(diethoxyphosphoryloxy)-(1-3)-benzotriazin-4 (3H)-one (DEPBT) as the major coupling reagent. The present work shows that: (i) the effects of linear peptide precursors on the cyclization are complex but some guidelines for choosing suitable precursor for cyclization could be considered; and (ii) DEPBT results in a higher cyclization yield compared with other coupling reagents. In addition, it was confirmed that peptides containing alternating D and L residues favor cyclization.     

Hydrazide reactive peptide tags for site-specific protein labeling

New site-specific protein labeling (SSPL) reactions for targeting-specific, short peptides could be useful for the real-time detection of proteins inside of living cells. One SSPL approach matches bioorthogonal reagents with complementary peptides. Here, hydrazide reactive peptides were selected from phage-displayed libraries using reaction-based selections. Selection conditions included washes of varying pH and treatment with NaCNBH(3) in order to specifically select reactive carbonyl-containing peptides. Selected peptides were fused to T4 lysozyme or synthesized on filter paper for colorimetric assays of the peptide-hydrazide interaction. A peptide-lysozyme protein fusion demonstrated specific, covalent labeling by the hydrazide reactive (HyRe) peptides in crude bacterial cell lysates, sufficient for the specific detection of an overexpressed protein fusion. Chemical synthesis of a short HyRe tag variant and subsequent reaction with two structurally distinct hydrazide probes produced covalent adducts observable by MALDI-TOF MS and MS/MS. Rather than isolating reactive carbonyl-containing peptides, we observed reaction with the N-terminal His of HyRe tag 114, amino acid sequence HKSNHSSKNRE, which attacks the hydrazide carbonyl at neutral pH. However, at the pH used during selection wash steps (<6.0), an alternative imine-containing product is formed that can be reduced with sodium cyanoborohydride. MSMS further reveals that this low pH product forms an adduct on Ser6. Further optimization of the novel bimolecular reaction described here could provide a useful tool for in vivo protein labeling and bioconjugate synthesis. The reported selection and screening methods could be widely applicable to the identification of peptides capable of other site-specific protein labeling reactions with bioorthogonal reagents.     

Easy parallel screening of reagent stability,quality control,and metrology in solid phase peptide synthesis (SPPS) and peptide couplings for microarrays

Evaluating the stability of coupling reagents, quality control (QC), and surface functionalization metrology are all critical to the production of high quality peptide microarrays. We describe a broadly applicable screening technique for evaluating the fidelity of solid phase peptide synthesis (SPPS), the stability of activation/coupling reagents, and a microarray surface metrology tool. This technique was used to assess the stability of the activation reagent 1‐{[1‐(Cyano‐2‐ethoxy‐2‐oxo‐ethylidenaminooxy)dimethylamino‐morpholinomethylene]}methaneaminiumHexafluorophosphate (COMU) (Sigma‐Aldrich, St. Louis, MO, USA) by SPPS of Leu‐Enkephalin (YGGFL) or the coupling of commercially synthesized YGGFL peptides to (3‐aminopropyl)triethyoxysilane‐modified glass surfaces. Coupling efficiency was quantitated by fluorescence signaling based on immunoreactivity of the YGGFL motif. It was concluded that COMU solutions should be prepared fresh and used within 5 h when stored at ~23 °C and not beyond 24 h if stored refrigerated, both in closed containers. Caveats to gauging COMU stability by absorption spectroscopy are discussed. Commercial YGGFL peptides needed independent QC, due to immunoreactivity variations for the same sequence synthesized by different vendors. This technique is useful in evaluating the stability of other activation/coupling reagents besides COMU and as a metrology tool for SPPS and peptide microarrays. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Fast conventional Fmoc solid-phase peptide synthesis with HCTU.

1H-Benzotriazolium 1-[bis(dimethyl-amino)methylene]-5-chloro-hexafluorophosphate (1-),3-oxide (HCTU) is a nontoxic, nonirritating and noncorrosive coupling reagent. Seven biologically active peptides (GHRP-6, (65-74)ACP, oxytocin, G-LHRH, C-peptide, hAmylin(1-37), and beta-amyloid(1-42)) were synthesized with reaction times reduced to deprotection times of 3 min or less and coupling times of 5 min or less using HCTU as the coupling reagent. Expensive coupling reagents or special techniques were not used. Total peptide synthesis times were dramatically reduced by as much as 42.5 h (1.8 days) without reducing the crude peptide purities. It was shown that HCTU can be used as an affordable, efficient coupling reagent for fast Fmoc solid-phase peptide synthesis.     

Novel mu-selective Met-enkephalinamide analogs with antagonist activity. Synthesis, receptor binding, analgesic properties and conformational studies

Four novel mu-selective peptide antagonists have been synthesized and examined for receptor binding, analgesic agonist and antagonist activity and energy conformational properties. These peptides were designed by analogy to results of molecular modeling of 3-phenyl piperidines which led to incorporating four modified tyrosine residues, m-Tyr, beta-methyl-m-Tyr, N-phenethyl-m-Tyr and alpha, beta-dimethyl-m-Tyr into D-Ala2-Met5-enkephalinamide. Peptides were synthesized by stepwise solution synthesis using an active ester coupling procedure. Receptor binding assays were performed on rat brain homogenates and data were analyzed by a modified version of the program LIGAND. Analgesic agonist and antagonist activity was evaluated by the mouse tail-flick test. Energy-optimized conformations were obtained using a program called Molecule-AIMS. The results demonstrate that relative ratios of in vivo agonist and antagonist potencies in D-Ala2-Met5-enkephalinamides can be modulated by chemical modification of the tyrosine residue. A shift in the phenolic-OH position from para to meta significantly enhances relative antagonist versus agonist activity; addition of a beta-CH3 group to the m-Tyr enhances mu-selectivity and leads to nearly equal agonist/antagonist activity. Energy conformational studies indicate that all analogs with high mu-receptor affinity examined have a common energy accessible B'II 2-3 turn conformation similar to that previously identified for high mu-affinity binding in peptides, lending further support to this candidate conformer. This conformer also has tyrosine side-chain angles which allowed total overlap with the amine and phenolic groups of a known structure of 3-(m-OH phenyl)-piperidine. This structural similarity together with the observation of mixed agonist antagonist activity in both types of opioids confirms the rationale upon which design of these peptides was based.     

Resin comparison and fast automated stepwise conventional synthesis of human SDF‐1α

Human SDF‐1α contains 68 amino acids and is a member of the chemokine family of peptides. This long peptide was synthesized stepwise using classical conditions in 101 h. The reaction times were then reduced to deprotection times of 2 × 2 min and coupling times of 2 × 2.5 min, resulting in a total synthesis time of 22 h. The effect of different resins, resin substitutions and deprotection reagents on the crude peptide purities was compared. A small portion of crude peptide was purified using an RP‐HPLC column and the mass of the final product was confirmed with MALDI‐TOF mass spectrometry. Copyright © 2008 European Peptide Society and John Wiley & Sons, Ltd.     

An NADH-tetrazolium-coupled sensitive assay for malate dehydrogenase in mitochondria and crude tissue homogenates

A sensitive spectrophotometric assay for determining mitochondrial malate dehydrogenase activity is described. The assay measures NADH production by coupling it to the reduction of 2-(p-iodophenyl)-3(p-nitrophenyl)-5-phenyl tetrazolium chloride (INT). Via an intermediate electron carrier, either phenazine methosulfate or lipoamide dehydrogenase, INT accepts electrons and is reduced to a red-colored formazan, which can be quantified by spectrophotometer at 500 nm. This assay uses only commercial reagents but gives a 2-5 fold (with lipoamide dehydrogenase) or 5-20 fold (with phenazine methosulfate) activity increase over currently available assays for pure enzyme in mitochondria isolated from human neuroblastoma cells, rat brain and liver, and crude homogenates of rat brain and liver. The assay can be easily performed with 96-well plate and less than 2.5 microg protein of isolated mitochondria or crude tissue homogenate. These results suggest that this assay is a simple, sensitive, stable and inexpensive method with wide application.     

Partial Asymmetric Synthesis in α-Alkylation of Enamine with Electrophilic Olefin

About 30 dipeptides and some tripeptides were led to new benzimidazole derivatives by incorporating their carboxyl groups into benzimidazole ring by the reaction with o-phenylenediamine. The ring closure to benzimidazole was well achieved by heating mildly at a moderate temperature in acetic acid.

Some benzimidazole derivatives of peptides had remarkable phytotoxicities.     

The development of highly efficient onium-type peptide coupling reagents based upon rational molecular design.

Novel and highly efficient immonium-, pyridinium- and thiazolium-type peptide coupling reagents, such as BOMI, BDMP, BPMP, BEP, FEP, BEPH, FEPH and BEMT, were developed by rational modifying of the molecular structures of commonly used uronium-type reagents. The high efficiency of these onium salts has been evaluated and proven by model reaction tests and the successful synthesis of various oligopeptides and biologically active peptides, both in solution and in the solid-phase, for example Leu-enkephalin, the pentapeptide moiety of Dolastatin 15 and the immunosuppressive undecapeptide cyclosporin O. Based upon these results, the relationship between the molecular structure and the capability of onium-type peptide coupling reagents was studied. A preliminary guideline for the molecular design of onium-type coupling reagents was developed.     

Synthesis and structure-activity relationships of elafin, an elastase-specific inhibitor.

Elafin, an elastase-specific inhibitor isolated from human skin, and its related peptides were synthesized by the solution procedure, and their inhibitory activities were measured against various enzymes. During the oxidative folding reactions of the reduced peptides, the ratio of the active product to the inactive product was varied by changing the concentration of guanidine HCl and the amount of redox reagents. The disulfide structures of fully active synthetic elafin and the inactive product were determined by amino acid analysis, gas-phase sequencing and mass spectrometry of their proteolytic fragments. The relationship between structure and inhibitory activities and/or the folding reaction was examined and the amino terminal part of the elafin molecule was found to have a great influence on the folding reactions, but not on the inhibitory activities.     

Active site directed inactivators of mouse submaxillary renin.

The following active site directed inactivators for the pressor enzyme renin were synthesized: L-alpha-bromo-isocaproyl(BIC)-Leu-Val-Tyr-Ser-OH, L-BIC-Val-Tyr-Ser-OH, L-BIC-Leu-Val-OCH3, L-BIC-Leu-Val-OH, L-BIC-Val-Tyr-NH2, L-BIC-Val-Tyr-OCH3, L-BIC-Val-Tyr-OH, L-BIC-Leu-OCH3, L-BIC-Val-OCH3, and L-BIC-OCH3. The rate of inactivation of mouse submaxillary gland renin by these reagents was studied under a variety of conditions. L-alpha-Bromoisocaproyl-Val-Tyr-Ser-OH and L-alpha-bromoisocaproyl-Leu-Val-Tyr-Ser-OH and L-alpha-bromoisocaproyl-Leu-Val-Tyr-Ser-OH were the most efficient inactivators followed by L-alpha-bromoisocaproyl-Val-Tyr-NH2. The rates of inactivation by the first two peptides were strongly dependent on pH, being most efficient at low pH, least efficient at pH near 5.6, and becoming efficient again at neutral pH. The rate of the inactivation by L-alpha-bromoisocaproyl-Val-Tyr-NH2, in which the C-terminal carboxyl group is blocked, was only slightly dependent on pH. Complete inactivation was achieved by these three reagents. The inactivation was accompanied by incorporation of a stoichiometric quantity of the radiolabeled reagents. Based on these findings it was concluded that the inactivators reacted with a carboxyl group(s) in the active site of the renin molecule to form an esteric linkage. These data also suggest that a carcoxyl group(s) may constitute part of the catalytically essential functional groups in renin action. D-alpha-Bromoisocaproyl derivatives of the various peptides mentioned above were also prepared. These compounds were much less active than the L isomers indicating that the inactivation by the L-alpha-bromoisocaproyl peptides was a specific reaction.     

Racemization studies in peptide synthesis using high-pressure liquid chromatography

We have synthesized the dipeptides benzoyl-l-phenylalanyl-l-alanyl benzyl ester and benzoyl-l-phenylalanyl-l-alanyl methyl ester by both the solid-phase and solution coupling methods. A variety of coupling reagents and solvents was employed. Each coupling reaction was analyzed by high-pressure liquid chromatography for extent of racemization. Baseline separations were achieved which allowed the direct, rapid, and reproducible determination of dipeptide diastereomers. Our successes in the separation of dipeptide diastereomers indicate the future value of applying high-pressure liquid chromatography to the separation of larger peptide diastereomers.     

(1H-benzotriazol-1-yloxy)-N,N-dimethylmethaniminium hexachloroantimonate (BOMI), a novel coupling reagent for solution and solid-phase peptide synthesis.

A HOBt-based immonium-type compound,(1H-benzotriazol-1-yloxy)-N,N-dimethyl methaniminium hexachloroantimonate (BOMI), was synthesized and successfully applied to the synthesis of various oligopeptides with good yields. The estimation of racemization and the influence of several reaction parameters such as solvents, bases and temperature were studied by HPLC using a model reaction. It was found that the reactivity of BOMI was much higher than that of HOBt-based phosphonium- and uronium-type coupling reagents. Moreover, its racemization was lower than that of other HOBt-derived coupling reagents. The effectiveness of BOMI was also demonstrated by the synthesis of Leu-enkephalin both in solution and in the solid-phase.     

Facile Synthesis of Benzimidazoles via Oxidative Cyclization of Acyclic Monoterpene Aldehyde with Diamines: Studies on Antimicrobial and in Vivo Evaluation of Zebrafish

Citral ( 1a ), a bioactive component of Cymbopogon citratus (lemongrass) could be isolated and semi-synthetic analogs synthesized with improved therapeutic properties. Herein we first report describes citral ( 1a ) as a primary material for the synthesis of benzimidazole derivatives between various o-phenylenediamines ( 2a – l ) in the presence of Diisopropylethylamine (DIPEA) as a commercially available environmentally benign base, ethanol as a green solvent and the yield of all benzimidazole derivatives ( 3a – l ) was between 68–76 %; The semi-synthetically prepared benzimidazole derivatives ( 3a – l ) were assessed for their anti-bacterial and anti-fungal properties. The benzimidazole compounds ( 3a – b , and 3g – j ) exhibit good anti-microbial activity. In addition, in silico study was carried out to determine the specific binding affinity of the diamine halogen substituted benzimidazole derivatives to the specific target proteins. In silico analysis revealed a high correlation between docking results and experimental results. Finally, benzimidazole demonstrated significant antibacterial and antifungal activity. Zebrafish embryos were subjected to In vivo toxicological test found that all of the benzimidazole compounds ( 3a – l ) were non-toxic and had low embryotoxicity after 96 h, with an LC₅₀ of 36.425 μg, which could facilitate the design of novel antimicrobial agents using a cost-effective method.     

Carboxy-terminal sequencing: formation and hydrolysis of C-terminal peptidylthiohydantoins

Proteins and peptides can be sequenced from the carboxy terminus with isothiocyanate reagents to produce amino acid thiohydantoin derivatives. Previous studies in our laboratory indicated that the use of trimethylsilyl isothiocyanate (TMS-ITC) as a coupling reagent significantly improved the yields and reaction conditions and reduced the number of complicating side products [Hawke et al. (1987) Anal. Biochem. 166, 298]. The present study further explores the conditions for formation of the peptidylthiohydantoins by TMS-ITC and examines the cleavage of these peptidylthiohydantoin derivatives into a shortened peptide and thiohydantoin amino acid derivative. Schizophrenia-related peptide (Thr-Val-Leu) was used as a model peptide and was treated with acetic anhydride and TMS-ITC at 50 degrees C for 30 min, and the peptidylthiohydantoin derivatives were isolated by reverse-phase HPLC and characterized by FAB-MS. The purified derivatives were subjected to a variety of cleavage conditions, and rate constants for hydrolysis were determined. Hydrolysis with acetohydroxamate as reported originally by Stark [(1968) Biochemistry 7, 1796] was found to give excellent cleavage of the terminal thiohydantoin amino acid, but also led to the formation of stable hydroxamate esters of the shortened peptide which are poorly suited for subsequent rounds of degradation. Hydrolysis with 2% aqueous triethylamine under mild conditions (1-5 min at 50 degrees C) was found to be more suitable for carboxy-terminal sequence analysis by the thiocyanate method. The shortened peptide, which could be isolated and subjected to a second round of degradation, and the released thiohydantoin amino acid are formed in good yield (90-100%). Several other small peptides containing 15 different C-terminal amino acid side chains were also investigated in order to examine any interfering reactions that might occur when these side chains are encountered in a stepwise degradation using the thiocyanate chemistry. Quantitative yields of peptidylthiohydantoins were obtained for all the amino acids examined with the following exceptions: low yields were obtained for C-terminal Glu or Thr, and no peptidylthiohydantoins were obtained for C-terminal Pro or Asp. Asparagine was found to form cyclic imides (64%) at the penultimate position (C-2) during hydrolysis of the peptidylthiohydantoins by 2% aqueous triethylamine. Cleavage of C-terminal Asn under these conditions led to the formation of the expected shortened peptide (69%), but also to the formation of a shortened peptide (31%) with a C-terminal amide. Problems with Glu and Thr could be solved by minimizing the reaction time with acetic anhydride.(ABSTRACT TRUNCATED AT 400 WORDS)     

Syntheses of Terpenes by the Condensation of Aliphatic Compounds

The condensation of mesityloxide with 4-diethylaminobutanone-(2) in the presence of potassium t-butoxide gave the products containing piperitenone together with isoxylitones, which were identified by gas chromatography and chemical methods. The effects of the combination of condensing agent, solvent, molar ratio of reagents and reaction temperature on the components in the condensation products were studied. It was found that piperitenone was able to obtain in an yield of 51% on the basis of 4-diethylaminobutanone-(2) by the use of Triton-B as a condensing agent. Also piperitenone can be isolated from the condensation products by the treatment with semicarbazide.     

Synthesis of bis- and tris-branched COOH-terminal pegylating reagents: conjugation to NH-terminal peptides.

In previous studies we reported an orthogonal protection scheme that was developed for the solution-phase synthesis of a family of bis- and tris-pegylating reagents which contain a free NH(2)-terminus. These pegylating reagents were coupled to the COOH-terminus of a model peptide. In the present study we report on the solution synthesis of a novel family of bis- and tris-pegylating reagents which contain a free COOH-terminus. To illustrate their general utility, conditions were developed for the coupling of these novel pegylating reagents to the NH(2)-function of a model pentapeptide. Taken together, our studies demonstrate that these pegylating reagents are well suited for conjugation to peptides and proteins that contain either free COOH- or NH(2)-functions. These reagents may have general utility in therapeutic development as branched pegylation has been shown to provide more effective protection of proteins from proteolysis by shielding the protein surface from approaching macromolecules.     

Mitochondrial permeabilization relies on BH3 ligands engaging multiple prosurvival Bcl-2 relatives, not Bak

The Bcl-2 family regulates apoptosis by controlling mitochondrial integrity. To clarify whether its prosurvival members function by sequestering their Bcl-2 homology 3 (BH3)-only ligands or their multidomain relatives Bak and Bax, we analyzed whether four prosurvival proteins differing in their ability to bind specific BH3 peptides or Bak could protect isolated mitochondria. Most BH3 peptides could induce temperature-dependent cytochrome c release, but permeabilization was prevented by Bcl-x(L), Bcl-w, Mcl-1, or BHRF1. However, their protection correlated with the ability to bind Bak rather than the added BH3 peptide and could be overcome only by BH3 peptides that bind directly to the appropriate prosurvival member. Mitochondria protected by both Bcl-x(L)-like and Mcl-1 proteins were disrupted only by BH3 peptides that engage both. BH3-only reagents freed Bak from Bcl-x(L) and Mcl-1 in mitochondrial and cell lysates. The findings support a model for the control of apoptosis in which certain prosurvival proteins sequester Bak/Bax, and BH3-only proteins must neutralize all protective prosurvival proteins to allow Bak/Bax to induce mitochondrial disruption.     

N-terminal α-amino group modification of antibodies using a site-selective click chemistry method

Site-specific conjugation of small molecules to antibody molecules is a promising strategy for generation of antibody-drug conjugates. In this report, we describe the successful synthesis of a novel bifunctional molecule, 6-(azidomethyl)-2-pyridinecarboxyaldehyde (6-AM-2-PCA), which was used for conjugation of small molecules to peptides and antibodies. We demonstrated that 6-AM-2-PCA selectively reacted with N-terminal amino groups of peptides and antibodies. In addition, the azide group of 6-AM-2-PCA enabled copper-free click chemistry coupling with dibenzocyclooctyne-containing reagents. Bifunctional 6-AM-2-PCA mediated site-specific conjugation without requiring genetic engineering of peptides or antibodies. A key advantage of 6-AM-2-PCA as a conjugation reagent is its ability to modify proteins in a single step under physiological conditions that are sufficiently moderate to retain protein function. Therefore, this new click chemistry-based method could be a useful complement to other conjugation methods.     

Design, synthesis, and antiviral evaluation of some polyhalogenated indole C-nucleosides

2,5, 6-Trichloro-1-(beta-D-ribofuranosyl)benzimidazole (TCRB), 2-bromo-5, 6-dichloro-1-(beta-D-ribofuranosyl)benzimidazole (BDCRB) and 2-benzylthio-5,6-dichloro-1-(beta-D-ribofuranosyl)benzimidazole (BTDCRB) are benzimidazole nucleosides that exhibit strong and selective anti-HCMV activity. Polyhalogenated indole C-nucleosides were prepared as 1-deaza analogs of the benzimidazole nucleosides TCRB and BDCRB. A mild Knoevenagel coupling reaction between an indol-2-thione and a ribofuranose derivative was developed for the synthesis of 2-benzylthio-5, 6-dichloro-3-(beta-D-ribofuranosyl)indole (12). 3-(beta-D-ribofuranosyl)-2,5,6-trichloroindole (16) was prepared from 12 in 4 steps. A Lewis acid-mediated glycosylation method was then developed to prepare the targeted 2-haloindole C-nucleoside 16 stereoselectively in four steps from the corresponding 2-haloindole aglycons. Only 12 was active against HCMV but it also was somewhat cytotoxic.