Analogues of deltorphin I containing conformationally restricted amino acids in position 2: structure and opioid activity

New analogues of deltorphin I (DT I, Tyr‐d ‐Ala‐Phe‐Asp‐Val‐Val‐Gly‐NH₂), with the d ‐Ala residue in position 2 replaced by α‐methyl‐β‐azido(amino, 1‐pyrrolidinyl, 1‐piperidinyl or 4‐morpholinyl)alanine, were synthesized by a combination of solid‐phase and solution methods. All ten new analogues were tested for receptor affinity and selectivity to μ‐ and δ‐opioid receptors. The affinity of analogues containing (R) or (S)‐α‐methyl‐β‐azidoalanine in position 2 to δ‐receptors strongly depended on the chirality of the α,α‐disubstituted residue. Peptide II , containing (S)‐α‐methyl‐β‐azidoalanine in position 2, displayed excellent δ‐receptor selectivity with its δ‐receptor affinity being only three times lower than that of DT I. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Influence of Mannosylation on Immunostimulating Activity of Adamant‐1‐yl Tripeptide

The mannosylated derivative of adamant‐1‐yl tripeptide (D ‐(Ad‐1‐yl)Gly‐L ‐Ala‐D ‐isoGln) was prepared to study the effects of mannosylation on adjuvant (immunostimulating) activity. Mannosylated adamant‐1‐yl tripeptide (Man‐OCH₂CH(Me)CO‐D ‐(Ad‐1‐yl)Gly‐L ‐Ala‐D ‐isoGln) is a non‐pyrogenic, H₂O‐soluble, and non‐toxic compound. Adjuvant activity of mannosylated adamantyl tripeptide was tested in the mouse model with ovalbumin as an antigen and in comparison to the parent tripeptide and peptidoglycan monomer (PGM, β‐D ‐GlcNAc‐(1→4)‐D ‐MurNAc‐L ‐Ala‐D ‐isoGln‐mesoDAP(εNH₂)‐D ‐Ala‐D ‐Ala), a well‐known effective adjuvant. The mannosylation of adamantyl tripeptide caused the amplification of its immunostimulating activity in such a way that it was comparable to that of PGM.     

Synthesis and Immunostimulating Properties of Novel Adamant‐1‐yl Tripeptides

The aim of this work was to prepare L ‐ and D ‐(adamant‐1‐yl)‐Gly‐L ‐Ala‐D ‐isoGln peptides in order to study their adjuvant (immunostimulating) activities. Adjuvant activity of adamant‐1‐yl tripeptides was tested in the mouse model using ovalbumin as an antigen and in comparison to the peptidoglycan monomer (PGM; β‐D ‐GlcNAc‐(1→4)‐D ‐MurNAc‐L ‐Ala‐D ‐isoGln‐mesoDAP(εNH₂)‐D ‐Ala‐D ‐Ala) and structurally related adamant‐2‐yl tripeptides.     

Vibrational and chiroptical spectroscopic characterization of γ‐turn model cyclic tetrapeptides containing two β‐Ala residues

The optical spectroscopic characterization of γ‐turns in solution is uncertain and their distinction from β‐turns is often difficult. This work reports systematic ECD and vibrational circular dichroism (VCD) spectroscopic studies on γ‐turn model cyclic tetrapeptides cyclo(Ala‐β‐Ala‐Pro‐β‐Ala) ( 1 ), cyclo(Pro‐β‐Ala‐Pro‐β‐Ala) ( 2 ) and cyclo(Ala‐β‐Ala‐Ala‐β‐Ala) ( 3 ). Conformational analysis performed at the 6‐31G(d)/B3LYP level of theory using an adequate PCM solvent model predicted one predominant conformer for 1‐3 , featuring two inverse γ‐turns. The ECD spectra in ACN of 1 and 2 are characterized by a negative n→π* band near 230 nm and a positive π→π* band below 200 nm with a long wavelength shoulder. The ECD spectra in TFE of 1‐3 show similar spectra with blue‐shifted bands. The VCD spectra in ACN‐d₃ of 1 and 2 show a +/?/+/? amide I sign pattern resulting from four uncoupled vibrations in the case of 1 and a sequence of two positive couplets in the case of 2 . A ?/+/+/? amide I VCD pattern was measured for 3 in TFE‐d₂. All three peptides give a positive couplet or couplet‐like feature (+/?) in the amide II region. VCD spectroscopy, in agreement with theoretical calculations revealed that low frequency amide I vibrations (at ～1630 cm^?1 or below) are indicative of a C₇ H‐bonded inverse γ‐turns with Pro in position 2, while γ‐turns encompassing Ala absorb at higher frequency (above 1645 cm^?1). Chirality, 2010. © 2010 Wiley‐Liss, Inc.     

Phylogenetic variation in glycosidases and glycanases acting on plant cell wall polysaccharides, and the detection of transglycosidase and trans-β-xylanase activities

Wall polysaccharide chemistry varies phylogenetically, suggesting a need for variation in wall enzymes. Although plants possess the genes for numerous putative enzymes acting on wall carbohydrates, the activities of the encoded proteins often remain conjectural. To explore phylogenetic differences in demonstrable enzyme activities, we extracted proteins from 57 rapidly growing plant organs with three extractants, and assayed their ability to act on six oligosaccharides ‘modelling’ selected cell‐wall polysaccharides. Based on reaction products, we successfully distinguished exo‐ and endo‐hydrolases and found high taxonomic variation in all hydrolases screened: β‐d ‐xylosidase, endo‐(1→4)‐β‐d ‐xylanase, β‐d ‐mannosidase, endo‐(1→4)‐β‐d ‐mannanase, α‐d ‐xylosidase, β‐d ‐galactosidase, α‐l ‐arabinosidase and α‐l ‐fucosidase. The results, as GHATAbase, a searchable compendium in Excel format, also provide a compilation for selecting rich sources of enzymes acting on wall carbohydrates. Four of the hydrolases were accompanied, sometimes exceeded, by transglycosylase activities, generating products larger than the substrate. For example, during β‐xylosidase assays on (1→4)‐β‐d ‐xylohexaose (Xyl₆), Marchantia, Selaginella and Equisetum extracts gave negligible free xylose but approximately equimolar Xyl₅ and Xyl₇, indicating trans‐β‐xylosidase activity, also found in onion, cereals, legumes and rape. The yield of Xyl₉ often exceeded that of Xyl_7–8, indicating that β‐xylanase was accompanied by an endotransglycosylase activity, here called trans‐β‐xylanase, catalysing the reaction 2Xyl₆→ Xyl₃ + Xyl₉. Similar evidence also revealed trans‐α‐xylosidase, trans‐α‐arabinosidase and trans‐α‐arabinanase activities acting on xyloglucan oligosaccharides and (1→5)‐α‐l ‐arabino‐oligosaccharides. In conclusion, diverse plants differ dramatically in extractable enzymes acting on wall carbohydrate, reflecting differences in wall polysaccharide composition. Besides glycosidase and glycanase activities, five new transglycosylase activities were detected. We propose that such activities function in the assembly and re‐structuring of the wall matrix.     

A quantitative strategy to detect changes in accessibility of protein regions to chemical modification on heterodimerization

We describe a method for studying quantitative changes in accessibility of surface lysine residues of the PB1 subunit of the influenza RNA polymerase as a result of association with the PA subunit to form a PB1‐PA heterodimer. Our method combines two established methods: (i) the chemical modification of surface lysine residues of native proteins by N‐hydroxysuccinimidobiotin (NHS‐biotin) and (ii) the stable isotope labeling of amino acids in cell culture (SILAC) followed by tryptic digestion and mass spectrometry. By linking the chemical modification with the SILAC methodology for the first time, we obtain quantitative data on chemical modification allowing subtle changes in accessibility to be described. Five regions in the PB1 monomer showed altered reactivity to NHS‐biotin when compared with the [PB1‐PA] heterodimer. Mutational analysis of residues in two such regions—at K265 and K481 of PB1, which were about three‐ and twofold, respectively, less accessible to biotinylation in the PB1‐PA heterodimer compared with the PB1 monomer, demonstrated that both K265 and K481 were crucial for polymerase function. This novel assay of quantitative profiling of biotinylation patterns (Q‐POP assay) highlights likely conformational changes at important functional sites, as observed here for PB1, and may provide information on protein–protein interaction interfaces. The Q‐POP assay should be a generally applicable approach and may detect novel functional sites suitable for targeting by drugs.     

The impact of β‐azido(or 1‐piperidinyl)methylamino acids in position 2 or 3 on biological activity and conformation of dermorphin analogues

The synthesis of new dermorphin analogues is described. The (R)‐alanine or phenylalanine residues of natural dermorphin were substituted by the corresponding α‐methyl‐β‐azidoalanine or α‐benzyl‐β‐azido(1‐piperidinyl)alanine residues. The potency and selectivity of the new analogues were evaluated by a competitive receptor binding assay in rat brain using [³H]DAMGO (a μ ligand) and [³H]DELT (a δ ligand). The most active analogue in this series, Tyr‐(R)‐Ala‐(R)‐α‐benzyl‐β‐azidoAla‐Gly‐Tyr‐Pro‐Ser‐NH₂ and its epimer were analysed by ¹H and ¹³C NMR spectroscopy and restrained molecular dynamics simulations. The dominant conformation of the investigated peptides depended on the absolute configuration around C^α in the α‐benzyl‐β‐azidoAla residue in position 3. The (R) configuration led to the formation of a type I β‐turn, whilst switching to the (S) configuration gave rise to an inverse β‐turn of type I′, followed by the formation of a very short β‐sheet. The selectivity of Tyr‐(R)‐Ala‐(R) and (S)‐α‐benzyl‐β‐azidoAla‐Gly‐Tyr‐Pro‐Ser‐NH₂ was shown to be very similar; nevertheless, the two analogues exhibited different conformational preferences. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

Cationic oligopeptides with the repeating sequence L‐lysyl‐L‐alanyl‐L‐alanine: conformational and thermal stability study using optical spectroscopic methods

The infrared (IR), vibrational circular dichroism (VCD), and electronic circular dichroism (ECD) spectra of short cationic sequential peptides (L ‐Lys‐L ‐Ala‐L ‐Ala)_n (n = 1, 2, and 3) were measured over a range of temperatures (20–90 °C) in aqueous solution at near‐neutral pH values in order to investigate their solution conformations and thermally induced conformational changes. VCD spectra of all three oligopeptides measured in the amide I′ region indicate the presence of extended helical polyproline II (PPII)‐like conformation at room temperature. UV‐ECD spectra confirmed this conclusion. Thus, the oligopeptides adopt a PPII‐like conformation, independent of the length of the peptide chain. However, the optimized dihedral angles ? and ψ are within the range ?82 to ?107° and 143–154°, respectively, and differ from the canonical PPII values. At elevated temperatures, the observed intensity and bandshape variations in the VCD and ECD spectra show that the PPII‐like conformation of the Lys‐Ala‐Ala sequence is still preferred, being in equilibrium with an unordered conformer at near‐neutral pH values within the range of temperatures from 20 to 90 °C. This finding was obtained from analysis of the temperature‐dependent spectra using the singular value decomposition method. The study presents KAA‐containing oligopeptides as conformationally stable models of biologically important cationic peptides and proteins. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Over‐expression of specific HvCslF cellulose synthase‐like genes in transgenic barley increases the levels of cell wall (1,3;1,4)‐β‐d‐glucans and alters their fine structure

Cell walls in commercially important cereals and grasses are characterized by the presence of (1,3;1,4)‐β‐d ‐glucans. These polysaccharides are beneficial constituents of human diets, where they can reduce the risk of hypercholesterolemia, type II diabetes, obesity and colorectal cancer. The biosynthesis of cell wall (1,3;1,4)‐β‐d ‐glucans in the Poaceae is mediated, in part at least, by the cellulose synthase‐like CslF family of genes. Over‐expression of the barley CslF6 gene under the control of an endosperm‐specific oat globulin promoter results in increases of more than 80% in (1,3;1,4)‐β‐d ‐glucan content in grain of transgenic barley. Analyses of (1,3;1,4)‐β‐d ‐glucan fine structure indicate that individual CslF enzymes might direct the synthesis of (1,3;1,4)‐β‐d ‐glucans with different structures. When expression of the CslF6 transgene is driven by the Pro35S promoter, the transgenic lines have up to sixfold higher levels of (1,3;1,4)‐β‐d ‐glucan in leaves, but similar levels as controls in the grain. Some transgenic lines of Pro35S:CslF4 also show increased levels of (1,3;1,4)‐β‐d ‐glucans in grain, but not in leaves. Thus, the effects of CslF genes on (1,3;1,4)‐β‐d ‐glucan levels are dependent not only on the promoter used, but also on the specific member of the CslF gene family that is inserted into the transgenic barley lines. Altering (1,3;1,4)‐β‐d ‐glucan levels in grain and vegetative tissues will have potential applications in human health, where (1,3;1,4)‐β‐d ‐glucans contribute to dietary fibre, and in tailoring the composition of biomass cell walls for the production of bioethanol from cereal crop residues and grasses.     

The crystal structure of the Y140F mutant of ADP-l-glycero-d-manno-heptose 6-epimerase bound to ADP-��-d-mannose suggests a one base mechanism

Bacteria synthesize a wide array of unusual carbohydrate molecules, which they use in a variety of ways. The carbohydrate L ‐glycero‐D ‐manno‐heptose is an important component of lipopolysaccharide and is synthesized in a complex series of enzymatic steps. One step involves the epimerization at the C6″ position converting ADP‐D ‐glycero‐D ‐manno‐heptose into ADP‐L ‐glycero‐D ‐manno‐heptose. The enzyme responsible is a member of the short chain dehydrogenase superfamily, known as ADP‐L ‐glycero‐D ‐manno‐heptose 6‐epimerase (AGME). The structure of the enzyme was known but the arrangement of the catalytic site with respect to the substrate is unclear. We now report the structure of AGME bound to a substrate mimic, ADP‐β‐D ‐mannose, which has the same stereochemical configuration as the substrate. The complex identifies the key residues and allows mechanistic insight into this novel enzyme.     

Crystallographic and mutational analyses of cystathionine β‐synthase in the H2S‐synthetic gene cluster in Lactobacillus plantarum

Cystathionine β‐synthase (CBS) catalyzes the formation of l ‐cystathionine from l ‐serine and l ‐homocysteine. The resulting l ‐cystathionine is decomposed into l ‐cysteine, ammonia, and α‐ketobutylic acid by cystathionine γ‐lyase (CGL). This reverse transsulfuration pathway, which is catalyzed by both enzymes, mainly occurs in eukaryotic cells. The eukaryotic CBS and CGL have recently been recognized as major physiological enzymes for the generation of hydrogen sulfide (H₂S). In some bacteria, including the plant‐derived lactic acid bacterium Lactobacillus plantarum, the CBS‐ and CGL‐encoding genes form a cluster in their genomes. Inactivation of these enzymes has been reported to suppress H₂S production in bacteria; interestingly, it has been shown that H₂S suppression increases their susceptibility to various antibiotics. In the present study, we characterized the enzymatic properties of the L. plantarum CBS, whose amino acid sequence displays a similarity with those of O‐acetyl‐l ‐serine sulfhydrylase (OASS) that catalyzes the generation of l ‐cysteine from O‐acetyl‐l ‐serine (l ‐OAS) and H₂S. The L. plantarum CBS shows l ‐OAS‐ and l ‐cysteine‐dependent CBS activities together with OASS activity. Especially, it catalyzes the formation of H₂S in the presence of l ‐cysteine and l ‐homocysteine, together with the formation of l ‐cystathionine. The high affinity toward l ‐cysteine as a first substrate and tendency to use l ‐homocysteine as a second substrate might be associated with its enzymatic ability to generate H₂S. Crystallographic and mutational analyses of CBS indicate that the Ala70 and Glu223 residues at the substrate binding pocket are important for the H₂S‐generating activity.     

Difference in the structures of alanine tri‐ and tetra‐peptides with antiparallel β‐sheet assessed by X‐ray diffraction,solid‐state NMR and chemical shift calculations by GIPAW

Alanine oligomers provide a key structure for silk fibers from spider and wild silkworms.We report on structural analysis of l ‐alanyl‐l ‐alanyl‐l ‐alanyl‐l ‐alanine (Ala)₄ with anti‐parallel (AP) β‐structures using X‐ray and solid‐state NMR. All of the Ala residues in the (Ala)₄ are in equivalent positions, whereas for alanine trimer (Ala)₃ there are two alternative locations in a unit cell as reported previously (Fawcett and Camerman, Acta Cryst., 1975, 31, 658–665). (Ala)₄ with AP β‐structure is more stable than AP‐(Ala)₃ due to formation of the stronger hydrogen bonds. The intermolecular structure of (Ala)₄ is also different from polyalanine fiber structure, indicating that the interchain arrangement of AP β‐structure changes with increasing alanine sequencelength. Furthermore the precise ¹H positions, which are usually inaccesible by X‐ray diffraction method, are determined by high resolution ¹H solid state NMR combined with the chemical shift calculations by the gauge‐including projector augmented wave method. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 13–20, 2014.     

Bifunctional glycosyltransferases catalyze both extension and termination of pectic galactan oligosaccharides

Pectins are the most complex polysaccharides of the plant cell wall. Based on the number of methylations, acetylations and glycosidic linkages present in their structures, it is estimated that up to 67 transferase activities are involved in pectin biosynthesis. Pectic galactans constitute a major part of pectin in the form of side‐chains of rhamnogalacturonan‐I. In Arabidopsis, galactan synthase 1 (GALS1) catalyzes the addition of galactose units from UDP‐Gal to growing β‐1,4‐galactan chains. However, the mechanisms for obtaining varying degrees of polymerization remain poorly understood. In this study, we show that AtGALS1 is bifunctional, catalyzing both the transfer of galactose from UDP‐α‐d ‐Gal and the transfer of an arabinopyranose from UDP‐β‐l ‐Ara_p to galactan chains. The two substrates share a similar structure, but UDP‐α‐d ‐Gal is the preferred substrate, with a 10‐fold higher affinity. Transfer of Ara_p to galactan prevents further addition of galactose residues, resulting in a lower degree of polymerization. We show that this dual activity occurs both in vitro and in vivo. The herein described bifunctionality of AtGALS1 may suggest that plants can produce the incredible structural diversity of polysaccharides without a dedicated glycosyltransferase for each glycosidic linkage.     

Effect of one D‐Leu residue on right‐handed helical ‐L‐Leu‐Aib‐ peptides in the crystal state

Four diastereomeric‐Leu‐Leu‐Aib‐Leu‐Leu‐Aib‐peptides, Boc‐D ‐Leu‐L ‐Leu‐Aib‐L ‐Leu‐L ‐Leu‐Aib‐OMe (1), Boc‐L ‐Leu‐D ‐Leu‐Aib‐L ‐Leu‐L ‐Leu‐Aib‐OMe (2), Boc‐L ‐Leu‐L ‐Leu‐Aib‐D ‐Leu‐L ‐Leu‐Aib‐OMe (3), and Boc‐L ‐Leu‐L ‐Leu‐Aib‐L ‐Leu‐D ‐Leu‐Aib‐OMe (4), were synthesized. The crystals of the four hexapeptides were characterized by X‐ray crystallographic analysis. Two diastereomeric hexapeptides 1 and 2 having D ‐Leu(1) or D ‐Leu(2) were folded into right‐handed (P) 3 ₁₀ ‐helical structures, while peptide 3 having D ‐Leu(4) was folded into a turn structure nucleated by type III′ and I$' \bf{\beta}$ ‐turns, and peptide 4 having D ‐Leu(5) was folded into a left‐handed (M) 3 ₁₀ ‐helical structure. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Conformational analysis of potent sweet taste ligands by NMR and computer simulations

We report the conformational analysis by ¹H‐nmr and computer simulations of five potent sweet molecules, N‐(3,3‐dimethylbutyl)‐L ‐aspartyl‐S‐(α‐methyl)phenylalanine methylester (1; 5000 times more potent than sucrose), L ‐aspartyl‐D ‐valine (S)‐α‐methoxycarbonylmethylbenzylamide (2; 1400 times more potent than sucrose), L ‐aspartyl‐D ‐valine α‐phenylcyclopentylamide (3; 1200 times more potent than sucrose), L ‐aspartyl‐D ‐α‐aminobutyric acid (S)‐α‐cyclohexylpropylamide (4; 2300 times more potent than sucrose), and L ‐aspartyl‐D ‐valine (R)‐α‐methylthiomethylbenzylamide (5; 3000 times more potent than sucrose). The “L‐shaped” structure, which we believe to be responsible for sweet taste, is accessible to all five sweet compounds in solution. This structure is characterized by a zwitterionic ring formed by the A‐H and B containing moieties located in the +y axis and by the hydrophobic group X pointing into the +x axis. Other accessible conformations of these flexible molecules are extended conformations with the A‐H and B containing moieties in the +y axis and the hydrophobic group X pointing in the −y axis and reversed L‐shaped structures with the hydrophobic group X projecting along the −x axis. The remarkable potency of the N‐alkylated compound 1 supports our recent hypothesis that a second hydrophobic binding domain in addition to interactions arising from the L‐shaped structure leads to an enhancement of sweetness potency. © 1999 John Wiley & Sons, Inc. Biopoly 49: 525–539, 1999     

Activation of pre-synaptic M-type K+ channels inhibits [3H]d-aspartate release by reducing Ca2+ entry through P/Q-type voltage-gated Ca2+channels

In this study, the functional consequences of the pharmacological modulation of the M‐current (I_KM) on cytoplasmic Ca²⁺ intracellular Ca²⁺concentration ([Ca²⁺]_i) changes and excitatory neurotransmitter release triggered by various stimuli from isolated rat cortical synaptosomes have been investigated. K_v7.2 immunoreactivity was identified in pre‐synaptic elements in cortical slices and isolated glutamatergic cortical synaptosomes. In cerebrocortical synaptosomes exposed to 20 mM [K⁺]_e, the I_KM activator retigabine (RT, 10 μM) inhibited [³H]d ‐aspartate ([³H]d ‐Asp) release and caused membrane hyperpolarization; both these effects were prevented by the I_KM blocker XE‐991 (20 μM). The I_KM activators RT (0.1–30 μM), flupirtine (10 μM) and BMS‐204352 (10 μM) inhibited 20 mM [K⁺]_e‐induced synaptosomal [Ca²⁺]_i increases; XE‐991 (20 μM) abolished RT‐induced inhibition of depolarization‐triggered [Ca²⁺]_i transients. The P/Q‐type voltage‐sensitive Ca²⁺channel (VSCC) blocker ω‐agatoxin IVA prevented RT‐induced inhibition of depolarization‐induced [Ca²⁺]_i increase and [³H]d ‐Asp release, whereas the N‐type blocker ω‐conotoxin GVIA failed to do so. Finally, 10 μM RT did not modify the increase of [Ca²⁺]_i and the resulting enhancement of [³H]d ‐Asp release induced by [Ca²⁺]_i mobilization from intracellular stores, or by store‐operated Ca²⁺channel activation. Collectively, the present data reveal that the pharmacological activation of I_KM regulates depolarization‐induced [³H]d ‐Asp release from cerebrocortical synaptosomes by selectively controlling the changes of [Ca²⁺]_i occurring through P/Q‐type VSCCs.     

Inhibition of NO Production by Grindelia argentina and Isolation of Three New Cytotoxic Saponins

A bioassay‐guided phytochemical analysis of the ethanolic extract of Grindelia argentina Deble & Oliveira ‐Deble (Asteraceae) allowed the isolation of a known flavone, hispidulin, and three new oleanane‐type saponins, 3‐O‐β‐D ‐xylopyranosyl‐(1→3)‐β‐D ‐glucopyranosyl‐2β,3β,16α,23‐tetrahydroxyolean‐12‐en‐28‐oic acid 28‐O‐β‐D ‐xylopyranosyl‐(1→2)‐β‐D ‐apiofuranosyl‐(1→3)‐β‐D ‐xylopyranosyl‐(1→3)‐α‐L ‐rhamnopyranosyl‐(1→2)‐α‐L ‐arabinopyranosyl ester ( 2 ), 3‐O‐β‐D ‐glucopyranosyl‐2β,3β,23‐trihydroxyolean‐12‐en‐28‐oic acid 28‐O‐β‐D ‐xylopyranosyl‐(1→2)‐β‐D ‐apiofuranosyl‐(1→3)‐β‐D ‐xylopyranosyl‐(1→3)‐α‐L ‐rhamnopyranosyl‐(1→2)‐α‐L ‐arabinopyranosyl ester, ( 3 ) and 3‐O‐β‐D ‐xylopyranosyl‐(1→3)‐β‐D ‐glucopyranosyl‐2β,3β,23‐trihydroxyolean‐12‐en‐28‐oic acid 28‐O‐β‐D ‐xylopyranosyl‐(1→2)‐β‐D ‐apiofuranosyl‐(1→3)‐β‐D ‐xylopyranosyl‐(1→3)‐α‐L ‐rhamnopyranosyl‐(1→2)‐α‐L ‐arabinopyranosyl ester ( 4 ), named grindeliosides A–C, respectively. Their structures were determined by extensive 1D‐ and 2D‐NMR experiments along with mass spectrometry and chemical evidence. The isolated compounds were evaluated for their inhibitory activities against LPS/IFN‐γ‐induced NO production in RAW 264.7 macrophages and for their cytotoxic activities against the human leukemic cell line CCRF‐CEM and MRC‐5 lung fibroblasts. Hispidulin markedly reduced LPS/IFN‐γ‐induced NO production (IC₅₀ 51.4 μM ), while grindeliosides A–C were found to be cytotoxic, with grindelioside C being the most active against both CCRF‐CEM (IC₅₀ 4.2±0.1 μM ) and MRC‐5 (IC₅₀ 4.5±0.1 μM ) cell lines.     

Controlling the helical screw sense of peptides with C‐terminal L‐valine

One chiral L ‐valine (L ‐Val) was inserted into the C‐terminal position of achiral peptide segments constructed from α‐aminoisobutyric acid (Aib) and α,β‐dehydrophenylalanine (Δ^ZPhe) residues. The IR, ¹H NMR and CD spectra indicated that the dominant conformations of the pentapeptide Boc‐Aib‐ΔPhe‐(Aib)₂‐L ‐Val‐NH‐Bn (3) and the hexapeptide Boc‐Aib‐ΔPhe‐(Aib)₃‐L ‐Val‐NH‐Bn (4) in solution were both right‐handed (P) 3₁₀‐helical structures. X‐ray crystallographic analyses of 3 and 4 revealed that only a right‐handed (P) 3₁₀‐helical structure was present in their crystalline states. The conformation of 4 was also studied by molecular‐mechanics calculations. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

DIFFERENCES IN POLYSACCHARIDE STRUCTURE BETWEEN CALCIFIED AND UNCALCIFIED SEGMENTS IN THE CORALLINE CALLIARTHRON CHEILOSPORIOIDES (CORALLINALES,RHODOPHYTA)1

The articulated coralline Calliarthron cheilosporioides Manza produces segmented fronds composed of calcified segments (intergenicula) separated by uncalcified joints (genicula), which allow fronds to bend and reorient under breaking waves in the wave‐swept intertidal zone. Genicula are formed when calcified cells decalcify and restructure to create flexible tissue. The present study has identified important differences in the main agaran disaccharidic repeating units [→3)‐β‐d ‐Galp (1→ 4)‐α‐l ‐Galp(1→] synthesized by genicular and intergenicular segments. Based on chemical and spectroscopical analyses, we report that genicular cells from C. cheilosporioides biosynthesize a highly methoxylated galactan at C‐6 position with low levels of branching with xylose side stubs on C‐6 of the [→3)‐β‐d ‐Galp (1→] units, whereas intergenicular segments produce xylogalactans with high levels of xylose and low levels of 6‐O‐methyl β‐d ‐Gal units. These data suggest that, during genicular development, xylosyl branched, 3‐linked β‐d ‐Galp units present in the xylogalactan backbones from intergenicular walls are mostly replaced by 6‐O‐methyl‐d‐ galactose units. We speculate that this structural shift is a consequence of a putative and specific methoxyl transferase that blocks the xylosylation on C‐6 of the 3‐linked β‐d ‐Galp units. Changes in galactan substitutions may contribute to the distinct mechanical properties of genicula and may lend insight into the calcification process in coralline algae.