Synthesis and In vitro Biological Activity of Cyclic Lipophilic χ-Conotoxin MrIA Analogues

The 13-residue peptide, χ-conotoxin MrIA extracted from the venom of Conus marmoreus, is a potent and selective inhibitor of the human noradrenaline transporter (NET). With the aim of improving its biophysical properties, chemical modifications were performed including the attachment of a lipophilic amino acid at the N-terminus and cyclisation of the peptide backbone with functionality introduced into the linker. All χ-conotoxin MrIA analogues were assembled on solid phase by highly optimised Boc chemistry and N- to C-cyclic analogues accessed by cysteine-mediated intramolecular native chemical ligation. In vitro biological activity at the human NET was evaluated by functional assays. All analogues inhibited the uptake of [³H]noradrenaline with comparable potencies to that of the native peptide, with one of the analogues, the linear N-terminal aminotetradecanoyl MrIA showing a 3-fold increase in potency (p < 0.05).     

Improved solid phase synthesis of luteinizing hormone releasing hormone analogues using 9-fluorenylmethyloxycarbonyl amino acid active esters and catalytic transfer hydrogenation with minimal side-chain protection and their biological activities

Using mainly 9-fluorenylmethyloxycarbonyl amino acid 2, 4, 5-trichlorophenyl esters in the presence of 1-hydroxybenzotriazole and the solid supportp-alkoxybenzyl alcohol resin, synthesis of luteinizing hormone releasing hormone analogues was carried out with minimal side-chain protection. Catalytic transfer hydrogenation was employed for removal of NO₂ and Z-groups from Arg and < Glu respectively avoiding the use of HF and this led to good yields. An aromatic, hydrophilic amino acid, D-(p-hydroxyphenyl) glycine was incorporated into luteinizing hormone releasing hormone molecule along with other modifications. The agonistic as well as antagonistic activities of all the peptides have been studied     

The use of N-urethane-protected N-carboxyanhydrides (UNCAs) in amino acid and peptide synthesis

N-Urethane-protected N-carboxyanhydrides (UNCAs) are very reactives. They have been successfully used in peptide synthesis, in both solution and solid phase. We have demonstrated that UNCAs are interesting starting materials for the synthesis of various amino acid derivatives. Chemoselective reduction of UNCAs with sodium borohydride led the corresponding N-protected β amino alcohols. Reaction of UNCAs with Meldrum's acid, followed by cyclisation, yielded enantiomerially pure tetramic acid derivatives. Diastereoselective reduction of tetramic acid derivatives produced (4S,5S)-N-alkoxycarbonyl-4-hydroxy-5-alkylpyrrolidin-2-ones derived from amino acids, which after hydrolysis yielded statine and statine analogues. Tetramic acid derivatives could also be obtained by reaction of UNCAs with benzyl ethyl followed by hydrogenolytic deprotection and decarboxylation. UNCAs also reacted with phosphoranes to produce the ketophosphorane in excellent yields. Subsequent oxidation with oxone or with [bis(acetoxy)-iodol]-benzene produced vicinal tricarbonyl derivatives. These reactions usually proceeded smoothly and with high yields.     

Synthesis and biological activity of some new leucine-enkephalin analogues

The opioid pentapeptide leucine-enkephalinamide and eleven of its analogues have been synthesised by the solid phase technique employing mostly 9-fluoroenylmethyloxycarbonyl amino acid active esters in the presence of 1-hydroxybenzotriazole. Both the conventional chloromethylated copolystyrene-2% divinylbenzene resin as well asp-alkoxybenzyl alcohol resin were employed and it was observed that yields were uniformly better with the latter resin. The analogues were made by affecting single or multiple replacements of amino acids involving positions 1,2 and 5. Some of the analogues were found to be more potent than morphine in the guinea pig ileum assay.     

Synthesis and Structure-activity Relationships of Amastatin Analogues,Inhibitors of Aminopeptidase A

Stereoisomers and analogues of amastatin, [(2S,3R)-3-amino-2-hydroxy-5-methylhexanoyl]-l-Val-l-Val-l-Asp, were synthesized and their inhibitory activities towards aminopeptidase A (AP-A) and other arylamidases tested. Among the four stereoisomers of a new amino acid residue in amastatin, the 2S stereoisomers exhibited strong activity. In a series of compounds in which the C-terminal amino acid of amastatin was substituted by other amino acids, the one containing Asp or Glu showed the strongest activity towards AP-A. In a series of compounds in which the second or third residue from the amino terminal of amastatin was substituted by other amino acids, the one containing hydrophobic amino acids showed strong activity. In the study of the relationship of the length of the peptide chain and inhibitory activity, the activity towards AP-A was seen to increase until the length of the peptide reached that of a tetrapeptide.     

Inhibition of Hog Liver Crystalline Quinolinate Phosphoribosyltransferase by Nucleotides,Quinolinate Analogues and Sulfhydryl Reagents

Effects of the precursors and intermediates of the NAD biosynthetic pathway, and of quinolinate analogues etc. on hog liver crystalline quinolinate phosphoribosyltransferase (an intermediary enzyme in the de novo NAD biosynthetic pathway) activity were investigated. The enzyme activity was inhibited by many kinds of nucleotides, phthalic acid and SH reagents. But amino acids, nicotinic acid and nicotinamide had practically no effect. The apparent inhibition by ATP removed by raising Mg²⁺ concentration. Phthalic acid was proved to be a competitive inhibitor to quinolinic acid. The Ki value for phthalic acid was calculated at 1.7 × 10^?4 m by a Dixon plot.     

Effects of Hydroxyproline and other Amino Acid Analogues on the Growth of Pea Root Segments

Changes in the lengths and growth rates of isolated 2–4 mm pea root segments, cultured in sucrose media under aseptic conditions, were paralleled by changes in invertase development and in chloride and leucine uptakes. The amino acid analogues o-, m- and p-fluorophenylalanine, azetidine-2-carboxylic acid and ethionine inhibited growth with corresponding changes in invertase activity and in chloride and leucine uptakes. In contrast hydroxyproline, which under the conditions used may be regarded as an analogue of proline, enhanced both the growth rate and duration of growth but had little effect on the several parameters of protein synthesis which were measured. No amino acid tested affected changes in growth, invertase activity or the uptake of chloride and leucine, but they prevented the effects of the corresponding analogues. The results show that although extension growth is dependent on continuous protein synthesis, only specific proteins, probably in the cell wall, play a key role in this process.     

Analysis of protegrin structure–activity relationships: the structural characteristics important for antimicrobial activity using smoothed amino acid sequence descriptors

Protegrin antimicrobial peptides (AMP) possess a high activity against a variety of microorganisms. In the present contribution, we analyse the structural requirements of protegrin analogues reported by Ostberg and Kaznessis (Peptides 2005; 26: 197) for having antimicrobial activity against several microbial species by using interpretable QSAR models. Models were carried out using multiple linear regression (MLR) combined with genetic algorithm (GA) and smoothed amino acid sequence properties were employed for characterizing the peptide dataset. The main advantage of smoothing process is the alteration of local amino acid properties by the properties of the amino acids in the closer neighbourhood. We report models encompassing different characteristics for describing the activities against different microbial species. Our results suggest the existence of specific mechanisms of action for protegrin analogues against different microbial species.     

A general method for preparation of N‐Boc‐protected or N‐Fmoc‐protected α,β‐didehydropeptide building blocks and their use in the solid‐phase peptide synthesis

N‐(tert‐butyloxycarbonyl) or N‐(9‐fluorenylmethoxycarbonyl) dipeptides with C‐terminal (Z)‐α,β‐didehydrophenylalanine (?^ZPhe), (Z)‐α,β‐didehydrotyrosine (?^ZTyr), (Z)‐α,β‐didehydrotryptophan (?^ZTrp), (Z)‐α,β‐didehydromethionine (?^ZMet), (Z)‐α,β‐didehydroleucine (?^ZLeu), and (Z/E)‐α,β‐didehydroisoleucine (?^Z/EIle) were synthesised from their saturated analogues via oxidation of intermediate 2,5‐disubstituted‐oxazol‐5‐(4H)‐ones (also known as azlactones) with pyridinium tribromide followed by opening of the produced unsaturated oxazol‐5‐(4H)‐one derivatives in organic‐aqueous solution with a catalytic amount of trifluoroacetic acid or by a basic hydrolysis. In all cases, a very strong preference for Z isomers of α,β‐didehydro‐α‐amino acid residues was observed except of the ΔIle, which was obtained as the equimolar mixture of Z and E isomers. Reasons for the (Z)‐stereoselectivity and the increased stability of the aromatic α,β‐didehydro‐α‐amino acid residue oxazol‐5‐(4H)‐ones over the corresponding aliphatic ones are also discussed. It is the first use of such a procedure to synthesise peptides with the C‐terminal unsaturated residues and a peptide with 2 consecutive ΔPhe residues. This approach is very effective especially in the synthesis of peptides with aliphatic α,β‐didehydro‐α‐amino acid residues that are difficult to obtain by other methods. It allowed the first synthesis of the ?Met residue. It is also more cost‐effective and less laborious than other synthesis protocols. The dipeptide building blocks obtained were used in the solid‐phase synthesis of model peptides on a polystyrene‐based solid support. Peptides containing aromatic α,β‐didehydro‐α‐amino acid residues were obtained with PyBOP or TBTU as a coupling agent with good yields and purities. In the case of aliphatic α,β‐didehydro‐α‐amino acid residues, a good efficiency was achieved only with DPPA as a coupling agent.     

Synthesis and Antibacterial Activity of Analogues of the N-Terminal Fragment of the Sarcotoxin IA Antimicrobial Peptide

Three 18-membered analogues of the N-terminal fragment of the sarcotoxin IA cationic antimicrobial peptide were synthesized by the solid phase method of peptide synthesis with the use of swellographic monitoring. The ability of these peptides to inhibit the growth of various bacteria in culture medium and their hemolytic activity in experiments on human erythrocytes were studied. The analogue completely corresponding to the N-terminal amino acid sequence of the natural sarcotoxin IA with the amide group on its C-terminus exhibited higher antibacterial activity. The presence of carboxyl group on the C-terminus or the substitution of Tyr for Trp2 resulted in a decrease in the antimicrobial activity of the peptide. Our results indicate that the amphiphilic N-terminal peptide corresponding to the 1–18 sequence of sarcotoxin IA involves the moieties responsible for the antimicrobial activity of the antibiotic.     

Variation of antimetabolite sensitivity with different carbon sources inBacillus subtilis

The susceptibility ofBacillus subtilis to amino acid analogues was found to be markedly influenced by the carbon source used in the test media. Thialysine inhibited the bacterium with a greater number of carbon sources than the other two analogues tested. 5-Hydroxylysine was inhibitory with glycerol, lactose,D-xylose,L-arabinose and soluble starch while ethionine showed toxicity with lactose,D-xylose andL-arabinose. None of these analogues were toxic at the levels tested whenD-galactose was used as carbon source. The bacterium was not susceptible to thialysine with glycerol, to 5-hydroxylysine withL-arabinose and to ethionine with lactose.     

Molecular Characterization of a Novel Family-46 Chitosanase from Pseudomonas sp. A-01

Pseudomonas sp. A-01, isolated as a strain with chitosan-degrading activity, produced a 28 kDa chitosanase. Following purification of the chitosanase (Cto1) and determination of its N-terminal amino acid sequence, the corresponding gene (cto1) was cloned by a reverse-genetic technique. The gene encoded a protein, composed of 266 amino acids, including a putative signal sequence (1-28), that showed an amino acid sequence similar to known family-46 chitosanases. Cto1 was successfully overproduced and was secreted by a Brevibacillus choshinensis transformant carrying the cto1 gene on expression plasmid vector pNCMO2. The purified recombinant Cto1 protein was stable at pH 5–8 and showed the best chitosan-hydrolyzing activity at pH 5. Replacement of two acidic amino acid residues, Glu23 and Asp41, which correspond to previously identified active centers in Streptomyces sp. N174 chitosanase, with Gln and Asn respectively caused a defect in the hydrolyzing activity of the enzyme.     

Design, Synthesis and Structure–Activity Relationship of New 109–116 Fragment Analogues of Antistasin and Ghilantens

A series of new low molecular weight peptide inhibitors, antistasin and ghilantens fragment analogues was designed and synthesized by manual solid phase peptide synthesis. These compounds only differ either by the amino acid placed in position 109 (different basic amino acids) and 115 position (Val or Ile) or 116 position Pro (as free acid or as amide). The anticoagulant activity of the different synthesized peptide mimetics was measured. Further the IC₅₀ was obtained by means of Activated Partial Thromboplastin Time measurement. Using Mihaelis–Menthen equation the mixed type of inhibition toward thrombin and Factor Xa is determined.     

Synthesis and preliminary conformational analysis of TOAC spin‐labeled analogues of the medium‐length peptaibiotic tylopeptin B

A set of analogues of the 14‐residue peptaibol tylopeptin B, containing the stable free‐radical 4‐amino‐1‐oxyl‐2,2,6,6,‐tetramethylpiperidine‐4‐carboxylic acid (TOAC) at one or two selected positions, was synthesized by the solid‐phase methodology. A solution conformational analysis performed by FTIR absorption and CD suggests that, in membrane‐mimicking solvents, the labeled tylopeptin B analogues preserve the helical propensity of the parent peptide, with a preference for the α‐helix or the 3₁₀‐helix type depending upon the nature of the solvent. In aqueous environment, the spin‐labeled analogues present a higher content of helical conformation as a consequence of the strong helix promoter effect of the conformationally constrained TOAC residue. We observed a progressive increase of the quenching effect of the nitroxyl radical on the fluorescence of the N‐terminal tryptophan as TOAC replaces the Aib residue at positions 13, 8, and 4, respectively. A membrane permeabilization assay performed on two selected analogues, TOAC⁸‐ and TOAC¹³‐tylopeptin B, showed that the labeled peptides exhibit membrane‐modifying properties comparable with those of the natural peptaibiotic. We conclude that our TOAC paramagnetic analogues of tylopeptin B are good models for a detailed ESR investigation of the mechanism of membrane permeabilization induced by medium‐length peptaibiotics. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

New Proctolin Analogues: Synthesis and Biological Investigation in Insects

Summary We have extended our work on structure/activity relationship studies of the neuropeptiden proctolin (H-Arg-Tyr-Leu-Pro-Thr-OH) by evaluating the effects of the following proctolin analogues: H-X¹-Tyr-Leu-Pro-Thr-OH, where X¹=d-Arg(I),N-Me-Arg (II), Can (III), Orn(di-Me) (IV), Orn (iPr) (V), Lys(N, N-di-Me) (VI), Lys(iPr) (VII), Lys(Nic) (VIII) andd-Lys(Nic) (IX). In analogues I–IX, the N-terminal Arg residue was replaced by basic amino acid derivatives with peptides containing amino acid residues with an isosteric system on the back side chain relative to Arg (compounds III, V and VI) orhomo-Arg (compound VII). Analogues I–IX were evaluated for myotropic activity on thein vitro heart preparation ofTenebrio molitor, whereas peptides II, V, and VII–IX were tested for contractile activity on the isolated foregut of locustSchistocerca gregaria. Peptide II and III showed full cardiotropic activity inT. molitor while peptides V and VII showed 40% and 15%, respectively, locust-gut contracting activity of proctolin.     

Side chain modifications of (indol-3-yl)glyoxamides as antitumor agents

New series of analogues of N-(pyridin-4-yl)-2-[1-(4-chlorobenzyl)-indol-3-yl]glyoxamide D-24851 were synthesized, characterized and tested for their in vitro anticancer properties. In the first series, an amino acid spacer was introduced in the glyoxamide chain of D-24851. In the second series, the glyoxamide chain was moved to positions 4 and 5 of indole skeleton. These new compounds were tested on four cancer cell lines (KB, SK-OV-3, NCI-H460 and SF-268), with promising activity for the glycine derivative.     

Conomarphins cause paralysis in mollusk: Critical and tunable structural elements for bioactivity

Two conomarphins were purified as the major component of the venom of Conus eburneus. Conomarphins Eb1 and Eb2 showed biological activity in the mollusk Pomacea padulosa, causing sluggishness and retraction of siphon, foot, and cephalic tentacles. To further probe the effects of conserved amino acids and posttranslational modifications in conomarphins, we prepared four synthetic analogues: conomarphin Eb1 Hyp10Pro, Hyp10Ala, d ‐Phe13Ala, and l ‐Phe13 variants. Structure‐activity relationship analysis indicated that d ‐Phe13 is critical to the biological activity of conomarphins. In contrast, amino acid changes at position 10 and removal of posttranslational modification in Hyp10Pro can be tolerated. The high expression level and observed mollusk activity of conomarphins may suggest their potential role as defensive arsenal of Conoidean snails against other predatory gastropods.     

Rutaceous alkaloids as models for the design of novel antitumor drugs

The chemical diversity of alkaloids in the Rutaceae is correlated with biosynthetic pathways involving various aromatic amino acid precursors, tyrosine, tryptophan, histidine, and anthranilic acid. The interest of rutaceous polyheteroaromatic alkaloids as models for the development of anticancer agents relies on their frequent ability to interact with DNA or with systems involved in the control of its topology, repair, and replication. Fagaronine and nitidine, from Zanthoxylum, demonstrate antileukemic activity, associated with topoisomerases inhibition. Evodiamine from Euodia rutaecarpa, displays antimetastatic properties. The pyranoacridone acronycine, from Sarcomelicope, exhibits antitumor activity against a broad spectrum of solid tumors. Development of synthetic analogues based on this latter natural product template followed the isolation of the unstable acronycine epoxide, which led to a hypothesis of bioactivation of acronycine by transformation of the 1,2-double bond into the corresponding oxirane. 1,2-Diacyloxy-1,2-dihydroacronycine derivatives exhibited antitumor properties, with a broadened spectrum of activity and an increased potency. The demonstration that acronycine interacted with DNA led to develop benzo[a], [b], and [c]acronycine analogs. Benzo[a] and [b] derivatives displayed significant antitumor activities. 1,2-Dihydroxy-1,2-dihydrobenzo[b]acronycine esters and diesters were active in human orthotopic models of cancers xenografted in nude mice. The activity of these compounds was correlated with their ability to give covalent adducts with DNA, involving reaction between the N-2 amino group of guanines and the ester group at the benzylic position of the drug. Cis-1,2-diacetoxy-1,2-dihydrobenzo[b]acronycine, currently developed under the code S23906-1, successfully underwent phase I and is currently under phase II clinical trials.     

Construction of minimum size cellulase (Cel5Z) from Pectobacterium chrysanthemi PY35 by removal of the C-terminal region

Pectobacterium chrysanthemi PY35 secretes the endoglucanase Cel5Z, an enzyme of the glycoside hydrolase family 5. Cel5Z is a 426 amino acid, signal peptide (SP)-containing protein composed of two domains: a large N-terminal catalytic domain (CD; 291 amino acids) and a small C-terminal cellulose binding domain (CBD; 62 amino acids). These two domains are separated by a 30 amino acid linker region (LR). A truncated cel5Z gene was constructed with the addition of a nonsense mutation that removes the C-terminal region of the protein. A truncated Cel5Z protein, consisting of 280 amino acid residues, functioned as a mature enzyme despite the absence of the SP, 11 amino acid CD, LR, and CBD region. In fact, this truncated Cel5Z protein showed an enzymatic activity 80% higher than that of full-length Cel5Z. However, cellulase activity was undetectable in mature Cel5Z proteins truncated to less than 280 amino acids.     

δ1-Pyrroline-5-carboxylate reductase as a new target for therapeutics: inhibition of the enzyme from Streptococcus pyogenes and effects in vivo

Compounds able to interfere with amino acid biosynthesis have the potential to inhibit cell growth. In both prokaryotic and eukaryotic microorganisms, unless an ornithine cyclodeaminase is present, the activity of δ¹-pyrroline-5-carboxylate (P5C) reductase is mandatory to proline production, and the enzyme inhibition should result in amino acid starvation, blocking in turn protein synthesis. The ability of some substituted derivatives of aminomethylenebisphosphonic acid and its analogues to interfere with the activity of the enzyme from the human pathogen Streptococcus pyogenes was investigated. Several compounds were able to suppress activity in the micromolar range of concentrations, with a mechanism of uncompetitive type with respect to the substrate P5C and non-competitive with respect to the electron donor NAD(P)H. The actual occurrence of enzyme inhibition in vivo was supported by the effects of the most active derivatives upon bacterial growth and free amino acid content.