L-proline Residue Research Articles

Structural dynamism of chiral sodium peraza-macrocycle complexes derived from cyclic peptoids.

A variety of cyclen and hexacyclen derivatives decorated with (S)-1-phenylethyl side chains or (S)-pyrrolidine units have been prepared via a reductive approach from the corresponding cyclic peptoids containing N-(S)-(1-phenylethyl)glycine and l-proline residues. Spectroscopic and DFT studies on their Na+ complexes show that point chirality and ring size play a crucial role in controlling the structural dynamism of 1,2-diaminoethylene units and pendant arms. The detection of highly symmetric C4- and C3-symmetric metalated species demonstrates that a full understanding of the relationship between the structure and conformational properties of peraza-macrocyclic metal complexes is possible.

Stereoselectivity of Proline/Cyclobutane Amino Acid-Containing Peptide Organocatalysts for Asymmetric Aldol Additions: A Rationale.

Several α,β,α- or α,γ,α-tripeptides, consisting of a central cyclobutane β- or γ-amino acid being flanked by two d- or l-proline residues, have been synthesized and tested as organocatalysts in asymmetric aldol additions. High yields and enantioselectivities have been achieved with α,γ,α-tripeptides, being superior to peptides containing a cyclobutane β-amino acid residue. This is probably due to their high rigidity, which hinders some of the peptide catalysts to adopt the proper active conformation. This reasoning correlates with the major conformation of the peptides in the ground state, as suggested by 1H NMR and computational calculations. The configuration of the aldol products is controlled by the proline chirality, and consequently, the R/S configuration of aldol products can be tuned by the use of either commercially available d- or l-proline. The enantioselectivity in the aldol reactions is reversed if the reactions are carried out in the presence of water or other protic solvents such as methanol. Spectroscopic and theoretical investigations revealed that this effect is not the consequence of conformational changes in the catalyst but rather caused by the participation of a water molecule in the rate determining transition state, in such a way that the preferential nucleophilic attack is oriented to the opposite enantiotopic aldehyde face.

Xylapeptide A, an Antibacterial Cyclopentapeptide with an Uncommon L-Pipecolinic Acid Moiety from the Associated Fungus Xylaria sp. (GDG-102)

Two new cyclopentapeptides, xylapeptide A (1) with an uncommon L-pipecolinic acid moiety, and xylapeptide B (2) having a common L-proline residue were identified from an associated fungus Xylaria sp. isolated from the Chinese medicinal plant Sophora tonkinensis. Their planar structures were elucidated by a comprehensive analysis of NMR and MS spectroscopic spectra. The absolute configurations were determined by Marfey’s method and single-crystal X-ray diffraction (Cu Kα) analysis. Xylapeptide A (1) is the first example of cyclopentapeptide with L-Pip of terrestrial origin and showed strong antibacterial activity against Bacillus subtilis and B. cereus with MIC value of 12.5 μg/mL.

Characterization of Enzymes Catalyzing Transformations of Cysteine S-Conjugated Intermediates in the Lincosamide Biosynthetic Pathway.

Lincosamides such as lincomycin A, celesticetin, and Bu-2545, constitute an important group of antibiotics. These natural products are characterized by a thiooctose linked to a l-proline residue, but they differ with regards to modifications of the thioacetal moiety, the pyrrolidine ring, and the octose core. Here we report that the pyridoxal 5'-phosphate-dependent enzyme CcbF (celesticetin biosynthetic pathway) is a decarboxylating deaminase that converts a cysteine S-conjugated intermediate into an aldehyde. In contrast, the homologous enzyme LmbF (lincomycin biosynthetic pathway) catalyzes C-S bond cleavage of the same intermediate to afford a thioglycoside. We show that Ccb4 and LmbG (downstream methyltransferases) convert the aldehyde and thiol intermediates into a variety of methylated lincosamide compounds including Bu-2545. The substrates used in these studies are the β-anomers of the natural substrates. The findings not only provide insight into how the biosynthetic pathway of lincosamide antibiotics can bifurcate to generate different lincosamides, but also reveal the promiscuity of the enzymes involved.

Prolyl-specific peptidases for applications in food protein hydrolysis.

Various food proteins including, e.g. gluten, collagen and casein are rich in L-proline residues. Due to the cyclic structure of proline, these proteins are well protected from enzymatic degradation by typical digestive enzymes. Proline-specific peptidases (PsP) belong to different families of hydrolases acting on peptide bonds (EC 3.4.x.x). They occur in various organisms including bacteria, fungi, plants and insects. Based on their biochemical characteristics, PsP type enzymes are further grouped into different subclasses of which prolyl aminopeptidases (EC 3.4.11.5, PAP), prolyl carboxypeptidases (EC 3.4.17.16, PCP) and prolyl oligopeptidases/prolyl endopeptidases (EC 3.4.21.26, POP/PEP) are of major interest for applications in food biotechnology. This mini review summarises the biochemical assays employed for these subclasses of PsP and their structural properties and the reaction mechanisms. A special focus was set on PsP derived from fungi and insects and important industrial applications in the field of food biotechnology. The degradation of gluten and collagen as well as the hydrolysis of bitter peptides are discussed.

An enzymatic method to estimate the content of L-hydroxyproline

Post-translational hydroxylation of the L-proline residue mainly occurs in collagen; therefore, the L-hydroxyprolines (L-Hyp) synthesized, including trans-4-hydroxy-L-proline (T4LHyp) and trans-3-hydroxy-L-proline (T3LHyp), are important markers for directly measuring the content of collagen in several biological samples. The most frequently used method to estimate the content of L-Hyp is high-performance liquid chromatography (HPLC), which is inconvenient. In the present study, we attempted to estimate the content of L-Hyp using coupling systems with metabolic enzymes of the T4LHyp (hydroxyproline 2-epimerase (HypE) and cis-4-hydroxy-D-proline dehydrogenase (HypDH)) and T3LHyp pathways (T3LHyp dehydratase (T3LHypD) and Δ1-pyrroline-2-carboxylate reductase (Pyr2CR)) from microorganisms. We constructed a functional expression system of recombinant HypDH with a heterooligomeric structure in Escherichia coli cells. Enzymological characterization revealed that the β-subunit acted as a catalytic subunit, and also that assembly with other subunit(s) improved the kinetics for cis-4-hydroxy-D-proline and thermostability. By using a spectrophotometric assay with different wavelengths, the contents of T4LHyp and T3LHyp were successfully estimated within the ranges of 0.004–1mM and 0.05–1mM, respectively, and were consistent with those determined by HPLC. This enzymatic method was used to measure the content of T4LHyp in the acid-hydrolysate of collagen, and blood plasma.

Genomic and Metabolomic Insights into the Natural Product Biosynthetic Diversity of a Feral-Hog-Associated Brevibacillus laterosporus Strain

Bacteria associated with mammals are a rich source of microbial biodiversity; however, little is known concerning the abilities of these microbes to generate secondary metabolites. This report focuses on a bacterium isolated from the ear of a feral hog from southwestern Oklahoma, USA. The bacterium was identified as a new strain (PE36) of Brevibacillus latersporus, which was shown via genomic analysis to contain a large number of gene clusters presumably involved in secondary metabolite biosynthesis. A scale-up culture of B. latersporus PE36 yielded three bioactive compounds that inhibited the growth of methicillin-resistant Staphylococcus aureus (basiliskamides A and B and 12-methyltetradecanoic acid). Further studies of the isolate's secondary metabolome provided both new (auripyrazine) and previously-described pyrazine-containing compounds. In addition, a new peptidic natural product (auriporcine) was purified that was determined to be composed of a polyketide unit, two L-proline residues, two D-leucine residues, one L-leucine residue, and a reduced L-phenylalanine (L-phenylalanol). An examination of the genome revealed two gene clusters that are likely responsible for generating the basiliskamides and auriporcine. These combined genomic and chemical studies confirm that new and unusual secondary metabolites can be obtained from the bacterial associates of wild mammals.

Conformation and cytotoxicity of a tetrapeptide constellated with alternative d- and l-proline

Proline containing peptides are highly important due to their natural abundance in various secondary structural elements like turns (β turn and γ turn etc.) in proteins. Here the conformation, cytotoxicity and structure of a unique tetrapeptide composed of alternative D- and L-proline residues are discussed. The peptide showed a polyproline II like conformation in dilute aqueous solution. The aqueous solution of the peptide self-assembled to form spheroidal oligomers with a diameter of ∼90 nm. The morphological features were confirmed by bright field confocal images, TEM analysis and AFM. The alternative D- and L-proline residues in the peptide showed toxicity towards cancer cell lines and ∼50% cell death was recorded against three different types of cancer cells (Neura 2a, HEK 293 and Hep G2).

Chirality responsive helical poly(phenylacetylene) bearing L‐proline pendants

A novel, optically active, cis-transoidal poly(phenylacetylene) bearing an L-proline residue as the pendant group (poly-1) was prepared by the polymerization of the corresponding monomer using a rhodium catalyst in water, and its chiroptical property was investigated using circular dichroism spectroscopy. Poly-1 showed intense Cotton effects in the UV-visible region of the polymer backbone in water, resulting from the prevailing one-handed helical conformation induced by the covalent-bonded chiral L-proline pendants and exhibited a unique helix-sense inversion in response to external, achiral, and chiral stimuli, such as the solvent and interactions with chiral small molecules. We found that poly-1 could enantioselectively trap 1,1'-2-binaphthol within its hydrophobic helical cavity inside the polymer in aqueous media and underwent an inversion of its helical sense in the presence of one of the enantiomers. The effect of the optical purity of 1,1'-2-binaphthol on the chiroptical properties of poly-1 was also investigated.

Switchable Perfomance of an l-Proline-Derived Basic Catalyst Controlled by Supramolecular Gelation

An L-proline-derived low molecular weight gelator forms gels in nitromethane and nitroethane and acts as a basic catalyst for the Henry nitroaldol reaction of these solvents with 4-nitrobenzaldehyde and 4-chlorobenzaldehyde. The reported catalyst is efficient only upon aggregation into self-assembled fibrillar networks. The formation of the gels is associated to a basicity boost of the L-proline residues. Gel dissociation blocks the catalytic efficiency for the nitroaldol reaction but enhances a reaction pathway leading to alkenes. Because of the reversible nature of supramolecular gels, subtle temperature changes allow for a reversible sol-gel transition associated to an activation of the catalyst. The catalytic gel from nitroethane is significantly more active than the one from nitromethane probably because of its different structure as revealed by X-ray diffraction and thermal stability studies. The results shown indicate that in solution the L-proline moiety catalyzes the reaction of nitroalkanes with aldehydes via iminium intermediates while efficient nitroaldol reactions are promoted in the gel phase through an ionic pair type mechanism. The fact that upon aggregation the amino acid-based molecule used as gelator plays both a structural (gel formation) and catalytic role is interesting for the point of view of life origin studies.

Preparation of l-proline based aeruginosin 298-A analogs: Optimization of the P 1-moiety

Aeruginosins are a family of naturally occurring oligopeptides that share a common bicyclic amino acid core structure. Many compounds in the family are inhibitors of serine proteases, such as thrombin and trypsin. Thrombin is an important enzyme in the blood coagulation cascade, and is a promising target for anticoagulant drug development. In order to understand the structure–activity relationship (SAR) and to find selective thrombin inhibitors, we synthesized a series of aeruginosin 298-A analogs, in which the P 2 bicyclic amino acid was replaced by a l-proline residue. The structure optimization was focused on modification of the P 1 position. In choosing the P 1 group, an effort was made to avoid using the highly basic guanidine groups present in nearly all naturally occurring aeruginosins. The synthesis and enzyme assays of these aeruginosin analogs against thrombin and trypsin are reported. We found that several compounds with neutral P 1 groups exhibit excellent selectivity over trypsin and good potency against thrombin. The SAR data of the P 1 groups obtained here can be used in preparing other thrombin inhibitors with better selectivity against trypsin.

Anticancer drug cis-4-hydroxy-L-proline: Correlation of preclinical toxicology with clinical parameters of liver function

cis-4-Hydroxy-L-proline (CHP) is being clinically evaluated as an anticancer drug. Since this compound targets the production of L-proline-rich proteins and critical L-proline residues, its impact on long-term cultures of human hepatocytes and toxicity in rats was studied to investigate possible effects on hepatic function, previously reported in rat hepatocytes. In the HEPAC2 human hepatocyte culture system, concentrations of CHP below 3.2 mg/ml had no significant effects on the release of lactate dehydrogenase (LDH), albumin, and urea. In rats, continuous administration of three different doses of CHP were tested for 28 days and resulted in signs of liver damage, as indicated by elevations of alanine aminotransferase (ALAT) and aspartate aminotransferase (ASAT) at a dose of 903 mg/kg, corresponding to a plasma concentration of approximately 200 µg/ml. Data from a clinical study of CHP in bladder and prostate cancer patients showed no adverse effects of administration of 8 g CHP/day, 4 days/week for 3 weeks in liver parameters ALAT, ASAT, γ-glutamyltransferase (γ-GT) and alkaline phosphatase (AP). In conclusion, the HEPAC2 human hepatocyte culture system correlates well with clinical results of a Phase II study of CHP, whereas a previous rat hepatocyte culture system predicted the compound would have toxic effects. The HEPAC2 system therefore constitutes a valuable tool for the preclinical screening of the hepatotoxicity of chemotherapeutic and other drugs, thereby reducing the need for experimental animals.

Kahalalides V-Y isolated from a Hawaiian collection of the sacoglossan mollusk Elysia rufescens.

Four new kahalalides, V (1), W (2), X (3), and Y (4), as well as six previously characterized kahalalides have been isolated from a two-year collection of the sacoglossan mollusk Elysia rufescens. Curiously, kahalalide B, previously isolated in high yield from E. rufescens, was found to be essentially absent from these collections despite identical collection sites and times with previous collections. In addition, kahalalide K, which to date has only been reported from Bryopsis sp., was found in this collection of E. rufescens, suggesting that the production of these metabolites could potentially be from a microbial association with the mollusk and algae, and this relationship is continuously evolving in response to changes in the environment and predation. The structures of new peptides have been established on the basis of extensive 1D and 2D NMR spectroscopic data analysis. Kahalalide V (1) was ascertained to be an acyclic derivative of kahalalide D (5), while kahalalide W (2) was determined to have a 4-hydroxy-L-proline residue instead of the proline in 5. The arginine residue of kahalalide X (3), an acyclic derivative of kahalalide C, was determined to have an L configuration. Kahalalide Y (4) was found to have an L-proline residue instead of the hydroxyproline in kahalalide K. It is clear from this collection of E. rufescens that the discovery of new kahalalide-related metabolites is still highly feasible.

Conformational analysis of α-helical polypeptide included l-proline residue by high-resolution solid-state NMR measurement and quantum chemical calculation

We challenged the problem about the stabilization mechanism of an α-helix formation for polypeptides containing l-proline (Pro) residue. We computed the optimized structure of α-helical poly( l-alanine) molecules including a Pro residue, H-(Ala) 8-Pro-(Ala) 9-OH, based on the molecular orbital calculation with density functional theory, B3LYP/6-31G(d) and the 13C and 15N chemical shift values based on the GIAO-CHF method with B3LYP/6-311G(d,p), respectively. It was found that two kinds of optimized structures, ‘Bent structure’ and ‘Included α-helix structure’, were preferred structures in H-(Ala) 8-Pro-(Ala) 9-OH. In addition, based on the precise 13C and 15N chemical shift data of the simple model, we successfully analyzed the secondary structure of well-defined synthetic polypeptide H-(Phe-Leu-Ala) 3-Phe C-Pro-Ala N-(Phe-Leu-Ala) 2-OH (FLA-11P), the secondary structure of which was proven to the ‘Included α-helix structure’.

Structure–activity relationships for the cardiotropic action of the Led‐NPF‐I peptide in the beetles Tenebrio molitor and Zophobas atratus

We have examined the effects of the Led-NPF-I peptide (Ala-Arg-Gly-Pro-Gln-Leu-Arg-Leu-Arg-Phe-amide) and a series of ten analogues on the heart contractile activity of Tenebrio molitor and Zophobas atratus, and the structure-activity relationships for cardioactive action of Led-NPF-I were established. A video microscopy technique and computer-based method of data acquisition and analysis were used to study the action of the peptides on continuously perfused heart preparations. Cardiac activity was progressively inhibited by Led-NPF-I when the peptide concentrations were increased from 10(-9) to 10(-5) M. Substitution of the L-proline residue at position 4 of the native peptide with hydroxyproline, valine or D-proline caused a loss of cardioinhibitory activity. Also, replacement of arginine residues at all three positions 2, 7 and 9 with another basic amino acid histidine, reduces cardioinhibitory action of Led-NPF-I. Some modifications of the C-terminal residues, as the Phe(4-NO2)-, Phe(4-NH2)- and Phe(4-NMe2)-analogues, resulted in agonistic peptides with biological activity similar to that of the native peptide. However, three other C-terminal analogues tested [Tyr10]-, [D-Phe10]-Led-NPF-I, and Ala-Arg-Gly-Pro-Gln-Leu-Arg-Leu-Arg-Phe-OH were inactive in the heart bioassay, which suggests that this end of the amino acid chain may play an important role in bioactivity and interaction of the native peptide with its receptor on the myocardium.

The Interaction between Polycationic Poly-Lysine Dendrimers and Charged and Neutral Fluorescent Probes

The interactions between polycationic poly-lysine dendrimers and hydrophobic fluorescent probes (anionic ANS and neutral Prodan) were studied. R121 and R131 dendrimers were not able to interact with anionic and neutral hydrophobic groups. R124 was able to interact with neutral and anionic hydrophobic fluorescent probes, however mainly through hydrophobic forces. Dendrimers R155 and R169 showed the maximal effects. The strongest interactions observed for R169 can be explained by intramolecular folding (stacking) of its two L-proline residues. Using double fluorescence titration technique for ANS probe allowed to receive such constant of binding and the number of binding centers: for R121, 1.8 x 10(3) (mol/l)(-1) and 1.07; for R124, 12.1 x 10(3) (mol/l)(-1) and 0.48; for R131, 4.7 x 10(3) (mol/l)(-1) and 0.48; for R155, 9.2 x 10(3) (mol/l)(-1) and 1.36; for R169, 39.6 x 10(3) (mol/l)(-1) and 0.97. Thus, neutral and anionic hydrophobic probes can be used for the fast preliminary screening of binding properties of newly synthesized polycationic dendrimers.

High-Performance Liquid Chromatographic Enantioseparation Based on Diastereomer Formation with New Fluorescent Chiral Quinoxalines

Two kinds of chiral 2,3-dimorpholinoquinoxalines bearing L-proline and N-bromoacetylproline residues at C-6 position were newly synthesized. The derivatization of (′)-naproxen, (′)-ibuprofen, and (′)-2-phenylpropionic acid with chiral quinoxalines afforded the diastereomers, which emitted an intense fluorescence. All the diastereomers were clearly separated within 16 min on reversed-phase (Finepak SIL C 1 8 S) HPLC equipped with a fluorescence detector. The detection limit of the derivatized product was estimated to be 5 pmol/10μL injection volume at S/N=5. Further, the quantum yield of the diastereomer was estimated to be 0.052 by using the relative quantum yield measurement with anthracene as a standard.

Proline‐Modified Porphyrin Catalysts for Enantioselective Epoxidations: Design, Synthesis, and Reactivity

AbstractThe syntheses of various strapped and ‘picket‐fence’ chiral porphyrins are described, and their reactivities towards the enantioselective epoxidation of alkenes are reported. Four L‐proline residues provide the chirality for the various meso‐substituted catalysts, which differ by either the spatial arrangement of the stereogenic centers or the nature and length of the straps. The resulting bridged structures possess four amide linkages in each strap, leading to highly rigid molecules with well‐defined geometries whereas the strapped Fe catalysts gave rise to only moderate enantioselectivities, the C2‐symmetrical ones being superior to the D2‐symmetrical compounds. The D2‐symmetrical ‘picket‐fence’ porphyrins were as selective as their strapped counterparts.

Proline–Glutamate Chimeras in Isopeptides. Synthesis and Biological Evaluation of Conformationally Restricted Glutathione Analogues

The two novel diastereoisomeric glutathione analogues 1 and 2 have been designed and synthesized by replacing the native γ-glutamylic moiety with the conformational rigid skeleton of cis- or trans-4-carboxy- l-proline residue. Both analogues have been obtained by following the solution phase peptide chemistry methodologies and final reduction of the corresponding disulfide forms 13 and 14. The two analogues 1 and 2 have been tested towards γ-glutamyltranspeptidase (γ-GT) and human glutathione S-transferase (hGST P1-1). Both analogues 1 and 2 are completely resistant to enzymatic degradation by γ-GT. The S-transferase utilizes the analogue 2 as a good substrate while is unable to bind the analogue 1.

Binding of D- and L-captopril inhibitors to metallo-beta-lactamase studied by polarizable molecular mechanics and quantum mechanics.

The bacterial Zn2+ metallo-beta-lactamase from B. fragilis is a zinc-enzyme with two potential metal ion binding sites. It cleaves the lactam ring of antibiotics, thus contributing to the acquired resistance of bacteria against antibiotics. The present study bears on the binuclear form of the enzyme. We compare several possible binding modes of captopril, a mercaptocarboxamide inhibitor of several zinc-metalloenzymes. Two diastereoisomers of captopril were considered, with either a D- or an L-proline residue. We have used the polarizable molecular mechanics procedure SIBFA (Sum of Interactions Between Fragments ab initio computed). Two beta-lactamase models were considered, encompassing 104 and 188 residues, respectively. The energy balances included the inter and intramolecular interaction energies as well as the contribution from solvation computed using a continuum reaction field procedure. The thiolate ion of the inhibitor is binding to both metal ions, expelling the bridging solvent molecule from the uncomplexed enzyme. Different competing binding modes of captopril were considered, either where the inhibitor binds in a monodentate mode to the zinc cations only with its thiolate ion, or in bidentate modes involving additional zinc binding by its carboxylate or ketone carbonyl groups. The additional coordination by the inhibitor's carboxylate or carbonyl group always occurs at the zinc ion, which is bound by a histidine, a cysteine, and an aspartate side chain. For both diastereomers, the energy balances favor monodentate binding of captopril via S-. The preference over bidentate binding is small. The interaction energies were recomputed in model sites restricted to captopril, the Zn2+ cations, and their coordinating end side chains from beta-lactamase (98 atoms). The interaction energies and their ranking among competing arrangements were consistent with those computed by ab initio HF and DFT procedures.