Microwave-assisted RCM for the synthesis of carbocyclic peptides.

Microwave irradiation dramatically improves the efficiency of ring closing metathesis (RCM) reactions of resin-attached peptides and the technology is illustrated by the highly selective synthesis of dicarba analogues of alpha-conotoxin IMI.     

Synthesis of a novel potent cyclic peptide MC4-ligand by ring-closing metathesis

The synthesis of a novel potent cyclic peptide MC4-ligand by ring-closing metathesis (RCM) is described. Based on the Ac-Nle-Gly-Lys-D-Phe-Arg-Trp-Gly-NH2-MC4 ligand, Ac-Nle-Alg-Lys-D-Phe-Arg-Trp-Alg-NH2 was designed and synthesized followed by cyclization using RCM. Both compounds are high affinity and selective MC4-R-agonists. The cyclic RCM-peptide was more potent in a rat-grooming assay.     

Synthesis,conformational analysis and biological properties of a dicarba derivative of the antimicrobial peptide,brevinin-1BYa

Brevinin-1BYa (FLPILASLAAKFGPKLFCLVTKKC), first isolated from skin secretions of the frog Rana boylii, displays broad-spectrum antimicrobial activity and potent haemolytic activity. This study investigates the effects on conformation and biological activity of replacement of the intramolecular disulphide bridge in the peptide by a non-reducible dicarba bond. Dicarba-brevinin-1BYa was prepared by microwave irradiation of [Agl¹⁸,Agl²⁴]-brevinin-1BYa (Agl = allylglycine) in the presence of a second generation Grubbs’ catalyst. Circular dichroism spectroscopy in 50% trifluoroethanol-water indicated that the degree of α-helicity of the dicarba derivative (22%) was less than that of brevinin-1BYa (27%) but comparable to that of the acyclic derivative [Ser¹⁸,Ser²⁴]-brevinin-1BYa (23%). Dicarba-brevinin-1BYa showed a two-fold increase in potency against reference strains of Escherichia coli, Staphylococcus aureus, and Candida albicans compared with the native peptide and displayed potent bactericidal activity against clinical isolates of methicillin-resistant S. aureus (MRSA) and multidrug-resistant Acinetobacter baumannii (MIC in the range 1–8 μM). Compared with brevinin-1BYa and [Ser¹⁸,Ser²⁴]-brevinin-1BYa, the dicarba derivative was associated with increased cytotoxicity against human erythrocytes (2.5-fold), MDA-MB-231 breast carcinoma cells (1.3-fold) and HepG2 hepatoma-derived cells (1.5-fold).     

A dicarba analog of beta-atrial natriuretic peptide (beta-ANP) inhibits guanosine 3',5'-cyclic monophosphate production induced by alpha-ANP in cultured rat vascular smooth muscle cells

The synthesis and biological properties are described of [Asu7,23']-beta-ANP-(7-28) (Asu, L-alpha-aminosuberic acid), a dicarba analog of beta-atrial natriuretic peptide (beta-ANP, an antiparallel dimer of human alpha-ANP with the chains linked by 7-23' and 7'-23 disulfide bonds). This Asu-analog (referred to as analog III) displaced 125I-alpha-ANP specifically bound to cultured rat vascular smooth muscle cells (VSMC) with an apparent Ki of 2.1 x 10(-8) M, but did not stimulate formation of intracellular cGMP at 10(-8) -10(-5) M. Analog III inhibited the alpha-ANP-stimulated cGMP production in VSMC competitively with a pA2 value of 7.45 and behaved as an antagonist of alpha-ANP in rat aorta smooth muscle relaxation. In addition, beta-ANP was also shown to inhibit the alpha-ANP-induced cGMP production in a dose-dependent manner. The mechanism of action of beta-ANP is also discussed.     

Synthesis of all-hydrocarbon stapled α-helical peptides by ring-closing olefin metathesis

This protocol provides a detailed procedure for the preparation of stapled α-helical peptides, which have proven their potential as useful molecular probes and as next-generation therapeutics. Two crucial features of this protocol are (i) the construction of peptide substrates containing hindered α-methyl, α-alkenyl amino acids and (ii) the ring-closing olefin metathesis (RCM) of the resulting resin-bound peptide substrates. The stapling systems described in this protocol, namely bridging one or two turns of an α-helix, are highly adaptable to most peptide sequences, resulting in favorable RCM kinetics, helix stabilization and promotion of cellular uptake.     

Application of ring-closing metathesis to Grb2 SH3 domain-binding peptides

Molecular processes depending on protein–protein interactions can use consensus recognition sequences that possess defined secondary structures. Left-handed polyproline II (PPII) helices are a class of secondary structure commonly involved with cellular signal transduction. However, unlike -helices, for which a substantial body of work exists regarding applications of ring-closing metathesis (RCM), there are few reports on the stabilization of PPII helices by RCM methodologies. The current study examined the effects of RCM macrocyclization on left-handed PPII helices involved with the SH3 domain-mediated binding of Sos1–Grb2. Starting with the Sos1-derived peptide “Ac-V1-P2-P3-P4-V5-P6-P7-R8-R9-R10-amide,” RCM macrocyclizations were conducted using alkenyl chains of varying lengths originating from the pyrrolidine rings of the Pro4 and Pro7 residues. The resulting macrocyclic peptides showed increased helicity as indicated by circular dichroism and enhanced abilities to block Grb2–Sos1 interactions in cell lysate pull-down assays. The synthetic approach may be useful in RCM macrocyclizations, where maintenance of proline integrity at both ring junctures is desired.     

The use of a novel reagent, Cu-FL, for determination of NO synthesis stimulated by the medicinal leech salivary cell secretion in human endothelial cell (HUVEC) and rat cardiomyocyte cultures

Using a novel NO-specific reagent, the complex of Cu²⁺ with a fluorescein derivative (Cu-FL), stimulation of NO production by the medicinal leech salivary cell secretion (SCS) has been demonstrated for the first time in cultures of human endothelial cells (HUVEC) and rat cardiomyocytes (RCM). NO was detected in the cells by fluorescent electronic microscopy and determined quantitatively in the cells and in the culture fluid by the fluorescence method. SCSstimulated NO synthesis in HUVEC but not in RCM cells was accompanied by NO release into the intercellular space thus determining its subsequent distribution. Localization of intracellular NO synthesis centers is presented and it is shown that the increase in NO levels during the SCS action on HUVEC and RCM is associated with the increase in the activity of eNOS/nNOS, but not iNOS. In the endothelial cells SCS-activated nitrosylation processes, estimated by the increase of nitrite-ion content in the culture medium. It is therefore important to use Cu-FL, rather than Griss-reagent, during the first hour of analysis of NO synthesis. It is possible that the NO-depended mechanism of the SCS action on endothelial cells may be a factor responsible for the positive effect of SCS during hirudotheraphy.     

Modulation of the antagonistic properties of an insulin mimetic peptide by disulfide bridge modifications

Insulin is a peptide responsible for regulating the metabolic homeostasis of the organism; it elicits its effects through binding to the transmembrane insulin receptor (IR). Insulin mimetics with agonistic or antagonistic effects toward the receptor are an exciting field of research and could find applications in treating diabetes or malignant diseases. We prepared five variants of a previously reported 20-amino acid insulin-mimicking peptide. These peptides differ from each other by the structure of the covalent bridge connecting positions 11 and 18. In addition to the peptide with a disulfide bridge, a derivative with a dicarba bridge and three derivatives with a 1,2,3-triazole differing from each other by the presence of sulfur or oxygen in their staples were prepared. The strongest binding to IR was exhibited by the peptide with a disulfide bridge. All other derivatives only weakly bound to IR, and a relationship between increasing bridge length and lower binding affinity can be inferred. Despite their nanomolar affinities, none of the prepared peptide mimetics was able to activate the insulin receptor even at high concentrations, but all mimetics were able to inhibit insulin-induced receptor activation. However, the receptor remained approximately 30% active even at the highest concentration of the agents; thus, the agents behave as partial antagonists. An interesting observation is that these mimetic peptides do not antagonize insulin action in proportion to their binding affinities. The compounds characterized in this study show that it is possible to modulate the functional properties of insulin receptor peptide ligands using disulfide mimetics.     

Erdosteine modulates radiocontrast‐induced hepatotoxicity in rat

It has been suggested that reactive oxygen species (ROS) plays an important role in radio contrast media (RCM)‐induced ischemia reperfusion tissue injury although antioxidants may have protective effects on the injury. We investigated the effects of erdosteine as an antioxidant agent on RCM‐induced liver toxicity in rats by evaluation of lipid peroxidation (as TBARS), catalase (CAT), superoxide dismutase (SOD), reduced glutathione (GSH) and glutathione peroxidase (GSH‐Px) values and histological evaluation. Twenty‐one rats were equally divided into three groups as follows: control, RCM, and RCM plus erdosteine. RCM was intraperitoneally administered for 1 day. Erdosteine was administered orally for 2 days after RCM administration. Liver samples were taken from the rats and they homogenized in a motor‐driven tissue homogenizer. TBARS levels were significantly (p < 0.005) higher in RCM group than in control although SOD activities significantly (p < 0.05) decreased in RCM group. TBARS levels were lower in RCM plus erdosteine group than in control although SOD activity and GSH level increased (p < 0.05) in liver as compared to RCM alone. Erdosteine showed also histopathological protection (p < 0.0001) against RCM induced hepatotoxicity. GSH‐Px and CAT activities were not statistically changed by the erdosteine. According to our results, it can be concluded that radiocontrast media can induce oxidative stress in liver as suggested by previous studies. Erdosteine seems to be protective agent on the radiocontrast media‐induced liver toxicity by inhibiting the production of ROS via the enzymatic antioxidant system. Copyright © 2009 John Wiley & Sons, Ltd.     

Modification of constrained peptides by ring-closing metathesis reaction

Ring-closing metathesis (RCM) with alpha,alpha-diallylglycyl peptides is shown to furnish alpha,alpha-cyclopentenylglycyl peptides as conformationally restrained analogues in the form of post-translational type peptide modification suitable for both peptidomimetic and combinatorial chemistry applications.     

Stapling mimics noncovalent interactions of γ-carboxyglutamates in conantokins, peptidic antagonists of N-methyl-D-aspartic acid receptors

Conantokins are short peptides derived from the venoms of marine cone snails that act as antagonists of the N-methyl-D-aspartate (NMDA) receptor family of excitatory glutamate receptors. These peptides contain γ-carboxyglutamic acid residues typically spaced at i,i+4 and/or i,i+7 intervals, which by chelating divalent cations induce and stabilize helical conformation of the peptide. Introduction of a dicarba bridge (or a staple) can covalently stabilize peptide helicity and improve its pharmacological properties. To test the hypothesis that stapling can effectively replace γ-carboxyglutamic acid residues in stabilizing the helical conformation of conantokins, we designed, synthesized, and characterized several stapled analogs of conantokin G (conG), with varying connectivities in terms of staple length and location along the face of the α-helix. NMR studies confirmed that the ring-closing metathesis reaction yielded a single product with the Z configuration of the olefinic bond. Based on circular dichroism and molecular modeling, the stapled analogs exhibited significantly enhanced helicity compared with the native peptide in a metal-free environment. Stapling i,i+4 was benign with respect to effects on in vitro and in vivo pharmacological properties. One analog, namely conG[11-15,S(i,i+4)S(8)], blocked NR2B-containing NMDA receptors with IC(50) = 0.7 μm and provided significant protection in the 6-Hz psychomotor model of pharmacoresistant epilepsy in mice. Remarkably, unlike native conG, conG[11-15,S(i,i+4)S(8)] produced no behavioral motor toxicity. Our results extend the applications of peptide stapling to helical peptides with extracellular targets and provide a means for engineering conantokins with improved pharmacological properties.     

Monitoring ring-closing metathesis: Limitations on the utility of H NMR analysis

The utility and limitations of ¹H NMR methods for monitoring the progress of ring-closing metathesis (RCM) reactions are examined. ¹H NMR analysis of RCM reactions that yield macrocyclic and medium-ring products can be more problematic than is generally recognized. Interpretation is complicated by the formation of oligomers with an NMR signature identical or closely similar to that of the diene precursor, the RCM target, or a composite of the two. DOSY-NMR can aid in resolving ambiguities in signal assignment, but reliable quantitation requires ancillary methods.     

Somatostatin subtype-2 receptor-targeted metal-based anticancer complexes

Conjugates of a dicarba analogue of octreotide, a potent somatostatin agonist whose receptors are overexpressed on tumor cells, with [PtCl(2)(dap)] (dap = 1-(carboxylic acid)-1,2-diaminoethane) (3), [(η(6)-bip)Os(4-CO(2)-pico)Cl] (bip = biphenyl, pico = picolinate) (4), [(η(6)-p-cym)RuCl(dap)](+) (p-cym = p-cymene) (5), and [(η(6)-p-cym)RuCl(imidazole-CO(2)H)(PPh(3))](+) (6), were synthesized by using a solid-phase approach. Conjugates 3-5 readily underwent hydrolysis and DNA binding, whereas conjugate 6 was inert to ligand substitution. NMR spectroscopy and molecular dynamics calculations showed that conjugate formation does not perturb the overall peptide structure. Only 6 exhibited antiproliferative activity in human tumor cells (IC(50) = 63 ± 2 μM in MCF-7 cells and IC(50) = 26 ± 3 μM in DU-145 cells) with active participation of somatostatin receptors in cellular uptake. Similar cytotoxic activity was found in a normal cell line (IC(50) = 45 ± 2.6 μM in CHO cells), which can be attributed to a similar level of expression of somatostatin subtype-2 receptor. These studies provide new insights into the effect of receptor-binding peptide conjugation on the activity of metal-based anticancer drugs, and demonstrate the potential of such hybrid compounds to target tumor cells specifically.     

Development and Placement of a Sorbent-Amended Thin Layer Sediment Cap in the Anacostia River

Incorporating materials into sediment caps that can sequester contaminants will greatly improve their ability to isolate contaminants in the underlying sediments from the rest of the aquatic environment. For highly sorptive media a thin layer (cm) may be sufficient, but accurately placing a thin layer (cm) of material over submerged contaminated sediment is difficult. A reactive core mat (RCM) was designed to accurately place a 1.25 cm thick sorbent (coke) layer in an engineered sediment cap. In April 2004, twelve 3.1 m × 31 m sections of RCM were placed in the Anacostia River, Washington, D.C., and overlain with a 15 cm layer of sand to secure it and provide a habitat for benthic organisms to colonize without compromising the integrity of the cap. Placement of the RCM did not cause significant sediment re-suspension or impact site hydrology. The RCM is an inexpensive and effective method to accurately deliver thin layers of difficult to place, high value, sorptive media into sediment caps. The approach can also be used to place granular reactive media that can degrade or mineralize contaminants.     

LRR conservation mapping to predict functional sites within protein leucine-rich repeat domains

Computational prediction of protein functional sites can be a critical first step for analysis of large or complex proteins. Contemporary methods often require several homologous sequences and/or a known protein structure, but these resources are not available for many proteins. Leucine-rich repeats (LRRs) are ligand interaction domains found in numerous proteins across all taxonomic kingdoms, including immune system receptors in plants and animals. We devised Repeat Conservation Mapping (RCM), a computational method that predicts functional sites of LRR domains. RCM utilizes two or more homologous sequences and a generic representation of the LRR structure to identify conserved or diversified patches of amino acids on the predicted surface of the LRR. RCM was validated using solved LRR+ligand structures from multiple taxa, identifying ligand interaction sites. RCM was then used for de novo dissection of two plant microbe-associated molecular pattern (MAMP) receptors, EF-TU RECEPTOR (EFR) and FLAGELLIN-SENSING 2 (FLS2). In vivo testing of Arabidopsis thaliana EFR and FLS2 receptors mutagenized at sites identified by RCM demonstrated previously unknown functional sites. The RCM predictions for EFR, FLS2 and a third plant LRR protein, PGIP, compared favorably to predictions from ODA (optimal docking area), Consurf, and PAML (positive selection) analyses, but RCM also made valid functional site predictions not available from these other bioinformatic approaches. RCM analyses can be conducted with any LRR-containing proteins at www.plantpath.wisc.edu/RCM, and the approach should be modifiable for use with other types of repeat protein domains.     

Quantification and visualization of coordination during non-cyclic upper extremity motion

There are many design challenges in creating at-home tele-monitoring systems that enable quantification and visualization of complex biomechanical behavior. One such challenge is robustly quantifying joint coordination in a way that is intuitive and supports clinical decision-making. This work defines a new measure of coordination called the relative coordination metric (RCM) and its accompanying normalization schemes. RCM enables quantification of coordination during non-constrained discrete motions. Here RCM is applied to a grasping task. Fifteen healthy participants performed a reach, grasp, transport, and release task with a cup and a pen. The measured joint angles were then time-normalized and the RCM time-series were calculated between the shoulder-elbow, shoulder-wrist, and elbow-wrist. RCM was normalized using four differing criteria: the selected joint degree of freedom, angular velocity, angular magnitude, and range of motion. Percent time spent in specified RCM ranges was used as a composite metric and was evaluated for each trial. RCM was found to vary based on: (1) chosen normalization scheme, (2) the stage within the task, (3) the object grasped, and (4) the trajectory of the motion. The RCM addresses some of the limitations of current measures of coordination because it is applicable to discrete motions, does not rely on cyclic repetition, and uses velocity-based measures. Future work will explore clinically relevant differences in the RCM as it is expanded to evaluate different tasks and patient populations.     

A straightforward route to enantiopure 2-substituted-3,4-dehydro-β-proline via ring closing metathesis

The synthesis of unusual cyclic amino acids, that may be envisaged as proline analogs, is an area of great interest for their potential applications as scaffolds for the design of bioactive peptidomimetics or units for the creation of novel foldamers. We have carried out the preparation of cyclic dehydro-β-amino acids starting from allylic carbonates via a two-step allylic amination/ring closing metathesis (RCM) protocol. The introduction of the allylamino moiety has been carried out either without a catalyst, through an S_N2′ reaction, or in the presence of iridium complexes. The backbone of the allylamino intermediates contains two unsaturations, thus suggesting that RCM could be a valuable tool for the preparation of dihydropyrrole scaffolds. A similar reaction has been already reported in the literature for racemic aromatic-substituted substrates, but no examples of enantiopure derivatives bearing aliphatic chains have been reported. The reaction was optimized by testing different Grubbs’ catalysts and carbamate nitrogen protecting groups. Moreover, in view of a future application of these dehydro-β-amino acids as central core of peptidomimetics, the malonate chain was also used to protect nitrogen prior to RCM.     

Synthesis of N-heterocyclic carbene ligands and derived ruthenium olefin metathesis catalysts

We describe the synthesis of commonly used free N-heterocyclic carbenes (NHCs), 1,3-bis-(2,4,6-trimethylphenyl)imidazol-2-ylidene (IMes) and 1,3-bis-(2,6-diisopropylphenyl)imidazol-2-ylidene (IPr), and of the two corresponding ruthenium-based metathesis complexes. The complex containing IMes was found to be highly efficient in macrocyclizations involving ring-closing metatheses (RCM), whereas the complex featuring the IPr ligand shows excellent activity in both RCM and cross metathesis because of its greater stability. The free carbenes IMes and IPr are isolated in four steps, with an overall yield of ～50%. They are then used to replace a labile phosphine in precatalysts belonging to two families of ruthenium-containing complexes, benzylidene and indenylidene types, respectively. Such complexes are isolated as analytically pure compounds with 77% and 95% yield. The total time for the synthesis of the free NHCs is 56 h, and incorporation in complexes requires an additional 4-5 h.     

Reflection contrast microscopy of ultrathin sections in immunocytochemical localization studies: a versatile technique bridging electron microscopy with light microscopy

Reflection contrast microscopy (RCM) of ultrathin sections was recently introduced as a sensitive technique for visualization with enhanced definition in immunogold histochemistry. Experience of using RCM as a major tool in immunocytochemical research in different fields is summarized, e.g. oncology, nephrology and embryology. The sensitive visualization of immunocytochemical labels, gold particles or peroxidase-diaminobenzidine deposits in or on ultrathin sections, by RCM instead of electron microscopy is demonstrated. RCM of ultrathin sections is an adequate light microscopical alternative for immunoelectron microscopy, since an overview of both label and tissue is obtained with a high image definition and high contrast of label. In the studies presented, RCM is shown to provide a better gradation in staining intensity and staining pattern than other light microscopical methods. Moreover, a precise localization of multiple labels is obtained with this method. Besides the applications shown, ultrathin section visualization by RCM is very useful for correlative light- and electron microscopical studies of fine structures. Commercially available fluorescence microscopes can be adapted for proper RCM functioning; an adaptation scheme and list of microscopes tested is provided.