Enzymatic synthesis of dehydroderivatives from proline-containing cyclic dipeptides and their effects toward cell division

We have previously isolated cyclo(L-Pro-L-Tyr) and cyclo(L-Phe-L-Pro) from an actinomycete by a novel enzymatic conversion-guided method. Their tetradehydro derivatives, cyclo(DeltaPro-DeltaTyr) and cyclo(DeltaPhe-DeltaPro), were enzymatically prepared. Neither of them inhibited cell division, in contrast to other tetradehydro cyclic dipeptides prepared previously. This result suggests that an NH proton in a diketopiperazine ring and/or conformation of the compound are important for the activity.     

Crystal structure and a twisted β-sheet conformation of the tripeptide L-leucyl-L-leucyl-L-leucine monohydrate trimethanol solvate: Conformation analysis of tripeptides

In order to test the helical preference of short oligo-L -leucines, we crystallized the tripeptide L -leucyl-L .-leucyl-L -leucine (LLL) and carried out x-ray diffraction studies of it (L -leucyl-L -leucyl-L leucine)₂. 3CH₃OH. H₂O, (C₃₉H₈₄N₆O₁₂). Crystallized in the monoclinic system, space group P2₁, cell parameters: a = 12.031(2), b = 15.578(3), c = 14.087(2) Å, α = 90°, β = 97.29(1)°, γ = 90°, V = 2618.6 ų. MW = 829.1, D_c = 1.051 gcm^?3. R index of 0.057 for 4213 reflections (λ_cukα = 1.5418 Å) > 2σ. LLL takes tip the β-sheet rather than a helical conformation in the crystalline stale. The three methanol molecules and the water molecule that constitute the solvent of crystallization form a network of hydrogen bonds to the LLL molecules and to one another. It is rather remarkable that though A and L have stronger helical preferences than G, neither AAA nor LLL form the crystalline helix but GAL does, indicating that the helical preferences depend on the sequence context. The residue L2 in molecule A and the residues L1 and L3 of molecule B do not show the preferred conformation for forming helices. Further, very remarkably. LLL exhibits a unique super secondary feature of the protein folding topology, namely the twisted β-sheet. Where as most short peptides show only the classical p-sheet conformation. Thai even the tripeptide LLL is able to exhibit the twisted β-sheet conformation, and with the correct left-handed twist this suggests that even very short peptide segments possess the ability to assume several of the characteristic topological features exhibited by proteins. An extensive review of tripeptide conformations has been carried out and some results of this study have been included here. © 1995 John Wiley & Sons, Inc.     

The biological activity of the histidine-containing diketopiperazines cyclo(His-Ala) and cyclo(His-Gly)

Two cyclic dipeptides, cyclo(His-Ala) and cyclo(His-Gly,) were synthesized from their linear counterparts and their structures elucidated using standard elucidation techniques. Molecular modeling and predictive NMR results indicated that the majority of energetically favourable conformers adopted a boat conformation with respect to the diketopiperazine ring. Cyclo(His-Ala), at concentrations of 100 microM inhibited the growth, in vitro, of various cancer cell lines, including HT-29, MCF-7 and HeLa carcinoma cells while cyclo(His-Gly) inhibited the growth of MCF-7 cells. While the antibacterial potential of these two compounds was limited, both cyclic dipeptides significantly inhibited the growth of C. albicans. Both compounds at a concentration of 100 microM resulted in a decrease in heart rate, coronary flow rate and left ventricular systolic pressure in the isolated rat heart. Inhibition of thrombin, amounting to a 63.3% and 36.7% reduction in the rate of fibrin formation, was noted for cyclo(His-Ala) and cyclo(His-Gly), respectively. While cyclo(His-Ala) showed no notable effects on platelet aggregation, cyclo(His-Gly) significantly inhibited both pathways tested with greatest effects on thrombin-induced platelet aggregation, yielding an IC(50) of 0.0662 mM (R(2)=0.989). The results of the anticancer and hematological studies indicate that histidine-containing diketopiperazines have potential as a novel group of cytotoxic agents with antithrombotic effects.     

Enzymatic Synthesis of Dehydroderivatives from Proline-Containing Cyclic Dipeptides and Their Effects toward Cell Division

We have previously isolated cyclo(L-Pro-L-Tyr) and cyclo(L-Phe-L-Pro) from an actinomycete by a novel enzymatic conversion-guided method. Their tetradehydro derivatives, cyclo(ΔPro-ΔTyr) and cyclo(ΔPhe-ΔPro), were enzymatically prepared. Neither of them inhibited cell division, in contrast to other tetradehydro cyclic dipeptides prepared previously. This result suggests that an NH proton in a diketopiperazine ring and/or conformation of the compound are important for the activity.     

Purification of an antifungal compound,cyclo(l-Pro-d-Leu) for cereals produced by Bacillus cereus subsp. thuringiensis associated with entomopathogenic nematode

Mold spoilage is the main cause of substantial economic loss in cereals and might also cause public health problems due to the production of mycotoxins. The aim of this study was to separate and purify and to identify antifungal compounds of bacterium associated with novel entomopathogenic nematode and check the antifungal property of identified compound in particular food model systems. The antifungal compound was purified using silica gel column chromatography, TLC and HPLC and its structure was elucidated using NMR (¹H NMR, ¹³C NMR, ¹H–¹H COSY, ¹H–¹³C HMBC), HRMS and Marfey's method. Based on the spectral data, the active compounds were identified as diketopiperazine [cyclo(l-Pro-d-Leu)]. The antifungal activity of cyclo(l-Pro-d-Leu) was studied by MIC and paper disk assay against Aspergillus flavus MTCC 277 and Aspergillus niger MTCC 282 and best MIC value of 8 μg/ml was recorded against A. flavus. Cyclo(l-Pro-d-Leu) strongly inhibit mycelia growth of fungus and thereby affecting aflatoxin production. To investigate the potential application of the cyclo(l-Pro-d-Leu) and to eliminate fungal spoilage in food and feed, soybean and peanut were used as models. White mycelia and dark/pale green spores of A. flavus were observed in the control soybeans after 2-day incubation. However the fungal growth was not observed in soybeans treated with cyclo(l-Pro-d-Leu). Almost the same result was observed for peanuts treated with cyclo(l-Pro-d-Leu) for A. niger. The cyclo(l-Pro-d-Leu) was nontoxic to two normal human cell lines (FS normal fibroblast and L231 lung epithelial) up to 200 μg/ml. Thus the diketopiperazine derivative identified in the study may be a promising alternative to chemical preservatives as a potential biopreservative which prevent fungal growth and mycotoxin formation in food and feed.     

The optical properties of alanine and proline diketopiperazines

The optical properties of the diketopiperazine chromophore of the cyclic dipeptides have been investigated as a function of molecular conformation. The rotatory strengths of L -alanyl–L -alanine diketopiperazine and L -prolyl–L -proline diketopiperazine have been calculated as a function of the angle of fold of the diketopiperazine ring. The results of these theoretical calculations have been compared with experimental circular dichroism and optical rotatory dispersion data. It is shown that the observed optical properties of these molecules can be explained only if their diketopiperazine rings are folded in opposite directions. The direction of fold is established for each molecule. In solution, the diketopiperazine ring of L -alanyl-L -alanine diketopiperazine is folded in the direction opposite to that found by X-ray diffraction analysis of crystals. It has been observed that the degree of conservatism of the π → π* couplet of L -propyl–L -proline diketopiperazine depends markedly upon the nature of the solvent that is used. In addition, a shoulder has been discovered in the CD spectrum of L -alanyl–L -alanine diketopiperazine, which may not be directly attributable to the n → π* and π → π* transitions of the peptide chromophores.     

Formation of Dendrolasin,Sesquirosefuran, Perillene and Rosefuran by Biomimetic Autooxidation

To estimate the steric distance between the bitter taste determinant sites in peptides, some cyclic dipeptides, amino acid anilides, amino acid cyclohexylamides, and benzoyl amino acids were synthesized and their tastes were evaluated. The diketopiperazine ring of cyclic dipeptides acted as a bitter taste determinant site due to its hydrophobicity. The steric distance between 2 sites was estimated as 4.1 Å from the molecule models of cyclic dipeptides composed of typical amino acids in the bitter peptides. Due to the hypothesis of two bitter taste determinant sites, which bind with the bitter taste receptor via a “binding unit” and a “stimulating unit,” a mechanism for the bitterness in peptides was postulated.     

Chronic Alcohol Consumption Increases Cyclo (His-Pro)-Like Immunoreactivity in the Rat Brain

Abstract: Administration of histidyl-proline diketopiperazine (cyclo (HisPro)) to rats attenuates ethanol-induced sleep. To understand the role played by cyclo (His-Pro) in the pathophysiology of prolonged alcohol consumption, we have measured the distribution of this peptide in brains of control and alcohol-treated rats. The data show that prolonged alcohol consumption increases the concentration of cyclo (His-Pro) in hypothalamic as well as extrahypothalamic brain. These changes may reflect a physiologic adaptation of the brain during alcohol consumption.     

Cyclo(L-prolyl-L-N-methylphenylalanyl). Conformation in solution and in the crystal.

Detailed analysis of the proton and carbon-13 NMR spectra of cyclo(L-prolyl-L-N-methylphenylalanyl) in chloroform and methanol in relation to its nonmethylated analog provided information on the conformation of the title compound in solution as well as on the effect of N-methylation and solvation. The X-ray structure of the title compound in the crystalline state showed the same conformational features as the solution structure. The phenyl group folds over the diketopiperazine ring which resembles a flattened half-chair. Both amide bonds are considerably nonplanar. The pyrrolidine ring of proline shows a strong pucker at the ring junction with the largest chi 5 value hitherto observed.     

3个六肽的胰岛素受体结合和体内生物活性

为了研究胰岛素受体结合部位的结构和功能,设计并用固相方法合成了３个六肽．在浓度大于１×１０３ｎｍｏｌ／Ｌ时,ｃｙｃｌｏ（Ｐｈｅ－Ｐｈｅ－Ｖａｌ－Ｌｅｕ－Ｔｙｒ－Ｇｌｙ）具有明显的胰岛素受体结合活力;Ｈ－Ｐｈｅ－Ｐｈｅ－Ｖａｌ－Ｌｅｕ－Ｔｙｒ－Ｇｌｙ－ＯＨ的这一活力则不明显;而Ｈ－Ｇｌｙ－Ｇｌｕ－Ａｒｇ－Ｇｌｙ－Ｐｈｅ－Ｐｈｅ－ＯＨ则增强胰岛素和其受体的亲和性．然而,它们都没有体内生物活性．这表明：环六肽部分模拟了胰岛素受体结合部位的空间构象;胰岛素受体结合部位的疏水性和其中的Ｂ２３Ｇｌｙ－Ｂ２４Ｐｈｅ－Ｂ２５Ｐｈｅ对胰岛素和其受体的结合起重要作用．     

Solution conformation and hydrolytic activity of cyclo(D-leu-L-His)

It has been reported previously that a cyclic dipeptide, cyclo(D -Leu-L -His), showed a high hydrolytic activity toward a hydrophobic ester, p-nitrophenyl laurate. In order to determine the reason for the high catalytic activity, the conformation of cyclo(D -Leu-L -His) in aqueous solution was investigated by nuclear magnetic resonance and circular dichroism spectroscopy and compared with the conformation of cyclo(L -Leu-L -His), which was nearly inactive in otherwise the same conditions for the hydrolysis. It was demonstrated that the spatial arrangement of the hydrophobic isobutyl group of the D -leucyl residue and of the nucleophilic imidazolyl group of the L -histidyl residue in cyclo(D -Leu-L -His) matches very well with the long acyl chain and the active ester function of p-nitrophenyl laurate. On the other hand, in cyclo(L -Leu-L -His) the hydrophobic and the nucleophilic pendant groups are too close with each other to cooperate intramolecularly for the hydrolysis. It was concluded that the different steric structures of the diastereomers can explain the large difference of the catalytic activities.     

Computational studies of ligand-receptor interactions in bitter taste receptors

Phenylthiocarbamide tastes intensely bitter to some individuals, but others find it completely tasteless. Recently, it was suggested that phenylthiocarbamide elicits bitter taste by interacting with a human G protein-coupled receptor (hTAS2R38) encoded by the PTC gene. The phenylthiocarbamide nontaster trait was linked to three single nucleotide polymorphisms occurring in the PTC gene. Using the crystal structure of bovine rhodopsin as template, we generated the 3D structure of hTAS2R38 bitter taste receptor. We were able to map on the receptor structure the amino acids affected by the genetic polymorphisms and to propose molecular functions for two of them that explained the emergence of the nontaster trait. We used molecular docking simulations to find that phenylthiocarbamide exhibited a higher affinity for the target receptor than the structurally similar molecule 6-n-propylthiouracil, in line with recent experimental studies. A 3D model was constructed for the hTAS2R16 bitter taste receptor as well, by applying the same protocol. We found that the recently published experimental ligand binding affinity data for this receptor correlated well with the binding scores obtained from our molecular docking calculations.     

Cyclic peptides. XIX. Synthesis, conformation, and biological activity of cyclo(-Pro-Val-Pro-Val-) with L-L-L-L and L-D-L-D sequences

cyclo(-L-Pro-L-Val-L-Pro-L-Val-) (1L) and cyclo(-L-Pro-D-Val-L-Pro-D-Val-) (1D) were synthesized by the conventional method for peptide synthesis. Conformations of 1L and 1D in solution were studied. Compound 1L has a cis-trans-cis-trans backbone conformation with C2 symmetry in CDCl3. This conformation is slightly different from that in crystalline state and in DMSO-d6 solution. Compound 1D has a cis-trans-cis-trans conformation in DMSO-d6 and an all-trans conformation in trifluoroethanol-d3. Compound 1L retarded stem-growth of rice seedlings and, in contrast, compound 1D promoted root-growth of rice seedlings.     

Computational Studies of Ligand-Receptor Interactions in Bitter Taste Receptors

Phenylthiocarbamide tastes intensely bitter to some individuals, but others find it completely tasteless. Recently, it was suggested that phenylthiocarbamide elicits bitter taste by interacting with a human G protein-coupled receptor (hTAS2R38) encoded by the PTC gene. The phenylthiocarbamide nontaster trait was linked to three single nucleotide polymorphisms occurring in the PTC gene. Using the crystal structure of bovine rhodopsin as template, we generated the 3D structure of hTAS2R38 bitter taste receptor. We were able to map on the receptor structure the amino acids affected by the genetic polymorphisms and to propose molecular functions for two of them that explained the emergence of the nontaster trait. We used molecular docking simulations to find that phenylthiocarbamide exhibited a higher affinity for the target receptor than the structurally similar molecule 6-n-propylthiouracil, in line with recent experimental studies. A 3D model was constructed for the hTAS2R16 bitter taste receptor as well, by applying the same protocol. We found that the recently published experimental ligand binding affinity data for this receptor correlated well with the binding scores obtained from our molecular docking calculations.     

2,5-Diketopiperazines: A New Class of Poly(ADP-ribose)polymerase Inhibitors

We show for the first time that natural 2,5-diketopiperazines (cyclic dipeptides) can suppress the activity of the important anticancer target poly(ADP-ribose)polymerase (PARP). Cyclo(L-Ala-L-Ala) and cyclo(L-Ala-D-Ala) can interact with the key residues of the PARP-1 active site, as demonstrated using docking and molecular dynamics simulations. One of the amide groups of cyclo(L-Ala-L-Ala) and cyclo(L-Ala-D-Ala) forms hydrogen bonds with the Gly863 residue, while the second amide group can form a hydrogen bond with the catalytic residue Glu988, and the side chain can make a hydrophobic contact with Ala898. Newly identified diketopiperazine inhibitors are promising basic structures for the design of more effective inhibitors of PARP family enzymes. The piperazine core with two chiral centers provides many opportunities for structural optimization.     

Synthesis and ex vivo profiling of chemically modified cytomegalovirus CMVpp65 epitopes.

The effect of substituting unnatural hydrophobic amino acids into the critical MHC binding residues of an HLA-A*0201-restricted cytomegalovirus CMVpp65 epitope, NLVPMVATV, has been investigated. A new set of peptides containing the amino acids tert-butyl glycine (Tgl), cyclohexyl glycine (Chg), neo-pentyl glycine (Npg), cyclohexyl alanine (Cha) and cyclo leucine (Cyl), at either position 2, to mimic Leu, or position 9, to mimic Val, have been synthesised. Immunological profiling using class I MHC stabilisation assays to assess MHC binding affinity, and enzyme-linked immunospot (ELISPOT) assays to assess the ability of the modified peptides to re-stimulate a specific cytotoxic T-lymphocyte (CTL) response, compared to the native epitope, have been performed. It was found that the majority of the unnatural substitutions resulted in a decrease in either HLA-A*0201 binding affinity or cytotoxic T-cell activity. However, the HLA-A*0201 binding affinity was unrelated to the ability to re-stimulate a T-cell response. Minimisation and molecular dynamics studies proved helpful in dissecting the ELISPOT responses. Two principal peptide binding modes were found by minimisation, designated kinked and straight. Peptides that bound in a kinked conformation were poor at re-stimulating a T-cell response. Of the peptides that bound in a straight conformation, molecular dynamics (MD) simulations revealed that those capable of re-stimulating the strongest responses had the greatest degree of flexibility (as determined by RMSD values across the MD simulation) around the P6 residue, one of the residues important for T-cell receptor recognition.     

Poly(L-leucyl-L-ornithyl-L-leucine) and its guanidization product: synthesis and antibacterial activity

The synthesis of poly(L -leucyl-L -ornithyl-L -leucine) and of poly(L -leucyl-L -arginyl-L -leucine) is reported. The antibacterial action of the two copolymers was checked by growth inhibition experiments on Staphylococcus aureus. The activity was found to be identical with that of a disordered 1:1 copolymer of L -ornithine and L -leucine. Conversion of 75% of the ornithine residues to arginine had no effect on the antibacterial activity.     

Structural analysis and the effect of cyclo(His–Pro) dipeptide on neurotoxins—a dynamics and density functional theory study

The switching propensity and maximum probability of occurrence of the side chain imidazole group in the dipeptide cyclo(His–Pro) (CHP) were studied by applying molecular dynamics simulations and density functional theory. The atomistic behaviour of CHP with the neurotoxins glutamate (E) and paraquat (Pq) were also explored; E and Pq engage in hydrogen bond formation with the diketopiperazine (DKP) ring of the dipeptide, with which E shows a profound interaction, as confirmed further by NH and CO stretching vibrational frequencies. The effect of CHP was found to be greater on E than on Pq neurotoxin. A ring puckering study indicated a twist boat conformation for the six-membered DKP ring. Molecular electrostatic potential (MESP) mapping was also used to explore the hydrogen bond interactions prevailing between the neurotoxins and the DKP ring. The results of this study reveal that the DKP ring of the dipeptide CHP can be expected to play a significant role in reducing effects such as oxidative stress and cell death caused by neurotoxins.     

Lack of effect of histidyl-proline diketopiperazine on basal level and TRH-induced response of cytosolic calcium concentration in rat lactotrophs.

Histidyl-proline diketopiperazine [cyclo(His-Pro)] has recently been shown to inhibit prolactin (PRL) secretion in vitro and in vivo. This peptide is well known as a metabolite of thyrotropin-releasing hormone (TRH), which is one of the endogenous secretagogues of PRL. In this study, we investigated the effect of cyclo (His-Pro) on the cytosolic Ca2+ concentration [[Ca2+]i) in cultured lactotrophs by using a lactotroph-enriched fraction separated from female rat pituicytes by centrifugal elutriation. TRH (10 nM) induced a rapid rise in [Ca2+]i in the lactotrophs, followed by a plateau phase of prolonged increase in [Ca2+]i. In contrast, the addition of 100 microM of cyclo (His-Pro) caused no changes in the basal level or the TRH-induced plateau response of [Ca2+]i. Although pretreatment with cyclo (His-Pro) tended to decrease the biphasic increase in [Ca2+]i induced by TRH, the inhibitory effect was not statistically significant. These results demonstrated that cyclo (His-Pro) has no effect on [Ca2+]i in lactotrophs, and does not affect the TRH-induced increase in [Ca2+]i, indicating that the inhibition of PRL secretion by cyclo (His-Pro) may be primarily mediated by other intracellular messengers such as cyclic nucleotides and secondarily involved in other inhibitory systems including that of dopamine.