Efficient, halide free synthesis of new, low cost ionic liquids: 1,3-dialkylimidazolium salts containing methyl- and ethyl-sulfate anions

New low-cost ionic liquids containing methyl- and ethyl-sulfate anions can be easily and efficiently prepared under ambient conditions by the reaction of 1-alkylimidazoles with dimethyl sulfate and diethyl sulfate. The preparation and characterization of a series of 1,3-dialkylimidazolium alkyl sulfate and 1,2,3-trialkylimidazolium alkyl sulfate salts are reported. 1,3-Dialkylimidazolium salts containing at least one non-methyl N-alkyl substituent are liquids at, or below room, temperature. Three salts were crystalline at room temperature, the single crystal X-ray structure of 1,3-dimethylimidazolium methyl sulfate was determined and shows the formation of discrete ribbons comprising of two anion-cation hydrogen-bonded chains linked via intra-chain hydrogen-bonding, but little, or no inter-ribbon hydrogen-bonding. The salts are stable, water soluble, inherently 'chloride-free', display an electrochemical window of greater than 4 V, and can be used as alternatives to the corresponding halide salts in metathesis reactions to prepare other ionic liquids including 1-butyl-3-methylimidazolium hexafluorophosphate.

Expression of the counter-regulatory peptide intermedin is augmented in the presence of oxidative stress in hypertrophied cardiomyocytes.

Background: Intermedin (IMD), a novel cardiac peptide related to adrenomedullin (AM), protects against myocardial ischemia-reperfusion injury and attenuates ventricular remodelling. IMD’s actions are mediated by a calcitonin receptor-like receptor in association with receptor activity modifying proteins (RAMPs 1-3). Aim/method: using the spontaneously hypertensive rat (SHR) and normotensive Wistar Kyoto (WKY) rat at 20 weeks of age, to examine (i) the presence of myocardial oxidative stress and concentric hypertrophy; (ii) expression of IMD, AM and receptor components. Results: In left and right ventricular cardiomyocytes from SHR vs. WKY cell width (26% left, 15% right) and mRNA expression of hypertrophic markers ANP (2.7 fold left, 2.7 fold right) and BNP (2.2 fold left, 2.0 fold right) were enhanced. In left ventricular cardiomyocytes only (i) oxidative stress was indicated by increased membrane protein carbonyl content (71%) and augmented production of O2- anion (64%); (ii) IMD (6.8 fold), RAMP1 (2.5 fold) and RAMP3 (2.0 fold) mRNA was increased while AM and RAMP2 mRNA was not altered; (iii) abundance of RAMP1 (by 48%), RAMP2 (by 41%) and RAMP3 (by 90%) monomers in cell membranes was decreased. Conclusion: robust augmentation of IMD expression in hypertrophied left ventricular cardiomyocytes indicates a prominent role for this counter-regulatory peptide in the adaptation of the SHR myocardium to the stresses imposed by chronic hypertension. The local concentration and action of IMD may be further enhanced by down-regulation of NEP within the left ventricle.

Ionic liquid as plasticizer for europium(III)-doped luminescent poly(methyl methacrylate) films

Flexible luminescent polymer films were obtained by doping europium(III) complexes in blends of poly(methyl methacrylate) (PMMA) and the ionic liquid 1-hexyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide, [C(6)mim][Tf2N]. Different europium(III) complexes have been incorporated in the polymer/ionic liquid matrix: [C(6)mim][Eu(nta)(4)], [C(6)mim][Eu(tta)(4)], [Eu(tta)(3)(phen)] and [choline](3)[Eu(dpa)(3)], where nta is 2-naphthoyltrifluoroacetonate, tta is 2-thenoyltrifluoroacetonate, phen is 1,10-phenanthroline, dpa is 2,6-pyridinedicarboxylate ( dipicolinate) and choline is the 2-hydroxyethyltrimethyl ammonium cation. Bright red photoluminescence was observed for all the films upon irradiation with ultraviolet radiation. The luminescent films have been investigated by high-resolution steady-state luminescence spectroscopy and by time-resolved measurements. The polymer films doped with beta-diketonate complexes are characterized by a very intense D-5(0) -> F-7(2) transition ( up to 15 times more intense than the D-5(0) -> F-7(1)) transition, whereas a marked feature of the PMMA films doped with [choline](3)[Eu(dpa)(3)] is the long lifetime of the D-5(0) excited state (1.8 ms).

Chemoenzymatic synthesis of the carbasugars carba-beta-L-galactopyranose, carba-beta-L-talopyranose and carba-alpha-L-talopyranose from methyl benzoate

The cis-dihydrodiol metabolite from methyl benzoate has been used as a synthetic precursor of carba-beta-L-galactopyranose, carba-beta-L-talopyranose and carba-alpha-L-talopyranose. The structures and absolute configurations of these carbasugars were determined by a combination of NMR spectroscopy, stereochemical correlation and X-ray crystallography.

Delivery of Methylene Blue and meso-tetra (N-methyl-4-pyridyl) porphine tetra tosylate from cross-linked poly(vinyl alcohol) hydrogels: A potential means of photodynamic therapy of infected wounds

Poly(vinyl alcohol)-borate complexes were evaluated as a potentially novel drug delivery platform suitable for in vivo use in photodynamic antimicrobial chemotherapy (PACT) of wound infections. An optimised formulation (8.0%w/w PVA, 2.0% w/w borax) was loaded with 1.0 mg ml(-1) of the photosensitisers Methylene Blue (MB) and meso-tetra (N-methyl-4-pyridyl) porphine tetra tosylate (TMP). Both drugs were released to yield receiver compartment concentrations (>5.0 mu g ml(-1)) found to be phototoxic to both planktonic and bicifilm-grown methicillin-resistant Staphylococcus aureus (MRSA), a common cause of wound infections in hospitals. Newborn calf serum, used to simulate the conditions prevalent in an exuding wound, did not adversely affect the properties of the hydrogels and had no significant effect on the rate of TMP-mediated photodynamic kill of MRSA, despite appreciably reducing the fluence rate of incident light. However, MB-mediated photodynamic kill of MRSA was significantly reduced in the presence of calf serum and when the clinical isolate was grown in a biofilm. Results support the contention that delivery of MB or TMP using gel-type vehicles as part of PACT could make a contribution to the photodynamic eradication of MRSA from infected wounds. (C) 2009 Elsevier B.V. All rights reserved.

Adrenomedullin Gene Delivery is Cardio-protective in a Model of Chronic Nitric Oxide Deficiency Combining Pressure Overload, Oxidative Stress and Cardiomyocyte Hypertrophy

BACKGROUND/AIMS:
Chronic inhibition of nitric oxide (NO) synthesis is associated with hypertension, myocardial ischemia, oxidative stress and hypertrophy; expression of the vasodilator peptide, adrenomedullin (AM) and its receptors is augmented in cardiomyocytes, indicating that the myocardial AM system may be activated in response to pressure loading and ischemic insult to serve a counter-regulatory, cardio-protective role. The study examined the hypothesis that oxidative stress and hypertrophic remodeling in NO-deficient cardiomyocytes are attenuated by adenoviral vector-mediated delivery of the human adrenomedullin (hAM) gene in vivo.

METHODS:
The NO synthesis inhibitor, N(G)-nitro-L-arginine methyl ester (L-NAME, 15mg . kg(-1) . day(-1)) was given to rats for 4 weeks following systemic administration via the tail vein of a single injection of either adenovirus harbouring hAM cDNA under the control of the cytomegalovirus promoter-enhancer (Ad.CMV-hAM-4F2), or for comparison, adenovirus alone (Ad.Null) or saline. Cardiomyocytes were subsequently isolated for assessment of the influence of each intervention on parameters of oxidative stress and hypertrophic remodelling.

RESULTS: Cardiomyocyte expression of the transgene persisted for > or =4 weeks following systemic administration of adenoviral vector. In L-NAME treated rats, relative to Ad.Null or saline administration, Ad.CMV-hAM-4F2 (i) reduced augmented cardiomyocyte membrane protein oxidation and mRNA expression of pro-oxidant (p22phox) and anti-oxidant (SOD-3, GPx) genes; (ii) attenuated increased cardiomyocyte width and mRNA expression of hypertrophic (sk-alpha-actin) and cardio-endocrine (ANP) genes; (iii) did not attenuate hypertension.

CONCLUSIONS: Adenoviral vector mediated delivery of hAM resulted in attenuation of myocardial oxidative stress and hypertrophic remodelling in the absence of blood pressure reduction in this model of chronic NO-deficiency. These findings are consistent with a direct cardio-protective action in the myocardium of locally-derived hAM which is not dependant on NO generation.

Peptide DV-28 amide: An inhibitor of bradykinin-induced arterial smooth muscle relaxation encoded by Bombina orientalis skin kininogen-2

Skin kininogens from bombinid toads encode an array of bradykinin-related peptides and one such kininogen from Bombina maxima also encodes the potent bradykinin B2-receptor antagonist, kinestatin. In order to determine if the skin secretion of the closely-related toad, Bombina orientalis, contained a bradykinin inhibitory peptide related to kinestatin, we screened reverse phase HPLC fractions of defensive skin secretion using a rat tail artery smooth muscle preparation. A fraction was located that inhibited bradykinin-induced relaxation of the preparation and this contained a peptide of 3198.5 Da as determined by MALDI-TOF MS. Automated Edman degradation of this peptide established the identity of a 28-mer as: DMYEIKGFKSAHGRPRVCPPGEQCPIWV, with a disulfide-bridge between Cys18 and Cys24 and an amidated C-terminal Val residue. Peptide DV-28 was found to correspond to residues 133–160 of skin pre-kininogen-2 of B. orientalis that also encodes two copies of (Thr6)-bradykinin. The C-terminal residue, Gly-161, of the precursor open-reading frame, acts as the C-terminal amide donor of mature DV-28. DV-28 amide thus represents a new class of bradykinin inhibitor peptide from amphibian skin secretion.

Phylloseptin-1 (PSN-1) from Phyllomedusa sauvagei skin secretion: a novel broad-spectrum antimicrobial peptide with antibiofilm activity

Amphibian skin secretions have proven to be rich sources of antimicrobial peptides that are proposed to be fundamental components of the innate immune system. As amphibian skin is a multi-functional organ playing, among other things, a crucial role in respiration, it has been deemed that a core biological role for such peptides is control of microbial flora on this surface. To date, however, antimicrobial efficacy has been universally determined by means of establishing minimum inhibitory concentrations (MICs) using planktonic organisms rather than those within a biofilm such as would occur on this exposed surface. Here we describe the identification and structural characterisation of a novel 19 amino acid residue antimicrobial peptide of the phylloseptin family, named PSN-1, from the skin secretion of the waxy monkey frog, Phyllomedusa sauvagei. PSN-1 displayed broad-spectrum activity against a range of planktonic organisms with a high potency (MIC 5 µM) against Staphylococcus aureus. In a specific bioassay with the same organism grown as a biofilm, the minimal biofilm eradication concentration (MBEC) was found to be of the same high potency (5 µM). The present data would suggest that evaluation of actions and potency of amphibian skin secretion antimicrobial peptides might best be achieved by evaluating MBEC rather than MIC using planktonic organisms and that data arising from such studies may have more biological relevance in reflecting the purpose for which they have evolved through natural selection.

Peptide DV-28 amide: Prototype of a novel class of bradykinin receptor antagonist isolated from the defensive skin secretion of bombinid toads

Kinestatin, isolated from the skin of the Chinese toad, Bombina maxima, was the first bradykinin B2 receptor antagonist identified in amphibians. Molecular cloning established that it is co-encoded with the bradykinin-related peptide, maximakinin, within one of several skin kininogens. To examine other species within the genus Bombina for the presence of structural homologues of kinestatin, we subjected skin secretion of the toad, Bombina orientalis, to HPLC fractionation with subsequent bioassay of fractions for antagonism of bradykinin activity using an isolated rat tail artery smooth muscle preparation. A single fraction was located that inhibited bradykinin-induced relaxation of rat arterial smooth muscle and MALDI-TOF analysis of this fraction revealed that it contained a single peptide of molecular mass 3198.5 Da. Further primary structural analysis of this peptide showed that it was a 28-mer with an N-terminal Asp (D) residue and a C-terminal Val (V) residue that was amidated. The peptide was named DV-28 amide in accordance with these primary structural attributes. Synthetic DV-28 amide replicated the observed bradykinin antagonistic effect within the smooth muscle bioassay in a dose-dependent manner. In addition, it was observed to inhibit the proliferation of human microvessel endothelial cells (HMECs) as assessed by MTT assay. Bioinformatic analysis revealed that DV-28 amide was, like kinestatin, co-encoded with a bradykinin receptor agonist on one of two skin kininogens identified in B. orientalis. DV-28 amide thus represents a novel class of bradykinin antagonist from skin secretions of bombinid toads that appear to be a rich source of such novel peptides.

Effects of the amphibian skin-derived bradykinin B2 receptor antagonist peptide, kinestatin, on the proliferation of human prostate cancer cell lines

Bradykinin and related peptides are found in the defensive skin secretions of many frogs and toads. While the physiological roles of bradykinin-related peptides in sub-mammalian vertebrates remains obscure, in mammals, including humans, canonical bradykinin mediates a multitude of biological effects including the proliferation of many types of cancer cell. Here we have examined the effect of the bradykinin B2 receptor antagonist peptide, kinestatin, originally isolated by our group from the skin secretion of the giant fire-bellied toad, Bombina maxima, on the proliferation of the human prostate cancer cell lines, PC3, DU175 and LnCAP. The bradykinin receptor status of all cell lines investigated was established through PCR amplification of transcripts encoding both B1 and B2 receptor subtypes. Following this demonstration, all cell lines were grown in the presence or absence of kinestatin and several additional bradykinin receptor antagonists of amphibian skin origin and the effects on proliferation of the cell lines was investigated using the MTT assay and by counting of the cells in individual wells of 96-well plates. All of the amphibian skin secretion-derived bradykinin receptor antagonists inhibited proliferation of all of the prostate cancer lines investigated in a dose-dependent manner. In addition, following incubation of peptides with each cell line and analysis of catabolites by mass spectrometry, it was found that bradykinin was highly labile and each antagonist was highly stable under the conditions employed. Bradykinin signalling pathways are thus worthy of further investigation in human prostate cancer cell lines and the evidence presented here would suggest the testing of efficacy in animal models of prostate cancer as a positive outcome could lead to a drug development programme for the treatment of this disease.

Molecular cloning of the helokinestatin/CNP precursor from a venom-derived cDNA library of the Mexican beaded lizard, Heloderma horridum, and identification of a novel encoded bradykinin-antagonist peptide, helokinestatin-6

Helokinestatins 1–5 represent a novel family of bradykinin antagonist peptides originally isolated from the venom of the Gila Monster, Heloderma suspectum. We found that they were encoded in tandem along with a single copy of C-type natriuretic peptide (CNP), by two different but almost identical biosynthetic precursors that were cloned from a venom-derived cDNA library. Here we have applied the same strategy to the venom of a related species, the Mexican beaded lizard, Heloderma horridum. Lyophilised venom was used as a surrogate tissue to generate a cDNA library that was interrogated with primers from the previous study and for reverse phase HPLC fractionation. The structure of a single helokinestatin precursor was obtained following sequencing of 20 different clones. The open-reading frame contained 196 amino acid residues, somewhat greater than the 177–178 residues of the corresponding helokinestatin precursors in H. suspectum. The reason for this difference in size was the insertion of an additional domain of 18 amino acid residues encoding an additional copy of helokinestatin-3. Helokinestatin-6 (GPPFNPPPFVDYEPR) was a novel peptide from this precursor identified in venom HPLC fractions. A synthetic replicate of this peptide antagonised the relaxation effect of bradykinin on rat arterial smooth muscle. The novel peptide family, the helokinestatins, have been shown to be present in the venom of H. horridum and to be encoded by a single precursor of different structure to those from H. suspectum. Studies such as this reveal the naturally-selected structures of bioactive peptides that have been optimised for purpose and provide the scientist with a natural analogue library for pharmacological investigation.

“Shotgun” cloning of skin peptide precursors from skin secretion-derived cDNA libraries of the Fujian large-headed frog, Limnonectes fujianensis

Amphibian skin secretions are rich sources of biologically-active peptides and several studies involving molecular cloning of their biosynthetic precursors have revealed that many exhibit highly-conserved domain architectures with an associated high degree of primary structural conservation of the signal peptides. This conservation of primary structure is reflected at the level of nucleotide sequence — a finding that has permitted our group to design primers to these sites facilitating “shotgun” cloning using cDNA libraries from uninvestigated species. Here we describe the results of such an approach using a skin secretion-derived cDNA library from the Fujian large-headed frog, Limnonectes fujianensis, a completely unstudied species. In over 50 clones studied by this approach, 12 were found to encode peptides of different primary structure. Representatives of 5 different families of antimicrobial peptides derived from the skins of ranid frogs were found and these were brevinin-1 (n = 3), the ranatuerin-2 (n = 3), esculentin-2 (n = 1), temporin (n = 1) and chensinin (n = 1). Three clones encoded peptides that were novel with no homologues present in contemporary on-line databases. These included two related 16-mer peptides, named peptides SC-16a and b, and an unrelated 24-mer, named peptide AG-24. Preliminary biological characterisation of SC-16a has demonstrated an antimicrobial activity against Gram-negative bacteria with a minimal inhibitory concentration of 35 µM with no observable haemolysis up to 200 µM. This finding may suggest that this peptide represents a novel class of antimicrobial with little effect on eukaryotic membranes.

A potent novel antimicrobial peptide related to caerin 1.12 from the skin secretion of the Australian frog, Litoria aurea

Skin secretions from Australian frogs of the genus Litoria have been extensively studied for many years and are known to contain a large array of antimicrobial peptides that often bear their specific names — caerins (L. caerulea), aureins (L. aurea), citropins (L. citropa) and maculatins (L. genimaculata) — and each group displays distinct primary structural attributes. During a systematic transcriptome cloning study using a cDNA library derived from skin secretion of L. aurea, a series of identical clones were identified that encoded a novel 25-mer antimicrobial peptide that displayed 92% structural identity with caerin 1.12 from L. caerulea, differing in amino acid sequence at only two positions — Arg for Gly at position 7 and Leu amide for Ser amide at the C-terminus. The novel peptide had conserved Pro residues at positions 15 and 19 that flank a flexible hinge region which previous studies have suggested are important for effective orientation of the two alpha-helices within the bacterial membrane resulting in lysis of cells. As the two substitutions in the novel peptide serve to increase both positive charge and hydrophobicity, we synthesised a replicate and determined its minimal inhibitory concentration (MIC) against Gram positive Staphylococcus aureus and Gram negative Escherichia coli. The MICs for these organisms were 3 µM and 4 µM, respectively, indicating a high potency and haemolysis was

GK-22 amide: a novel myotropic peptide from the skin secretion of the bamboo leaf odorous frog, Odorrana versabilis

The skin secretions produced by many amphibians are formidable chemical/biological weapons deployed as a defence against predators. Bioactive peptides are often the predominant class of biochemical within these secretions and the inventory of such remains incomplete with each individual taxon producing unique cocktails contained within which are some signature peptides, such as bradykinins and tachykinins. These secretions have been the source of many peptides subsequently found to have structural homologues in vertebrate neuroendocrine systems (bombesin/GRP; sauvagine/CRF; caerulein/CCK) and vice versa (bradykinin, CGRP, NMU). They are thus unequivocally intriguing resources for novel bioactive peptide discovery. Here we describe a novel 22-mer amidated peptide, named GK-22 amide (N-terminal Gly (G) and C-terminal Lys (K) amide) with an internal disulphide bridge between Cys (C) 11 and 20 from the skin secretion of Odorrana versabilis. Molecular cloning indicated that it is encoded as a single copy on a biosynthetic precursor of 59 amino acid residues consisting of a signal peptide, an acidic amino acid residue-rich spacer domain and a mature peptide encoding domain flanked N-terminally by a classical -Lys-Arg- (KR) propeptide convertase processing site and C-terminally by a Gly (G) residue amide donor. A synthetic replicate of this peptide produced potent and dose-dependent contraction of the smooth muscle of rat urinary bladder. GK-22 amide thus represents the prototype of a novel class of myotropic peptide from amphibian skin and its discovery illustrates the continuing potential of this resource to this end.