The use of 2,2′‐dithiobis(5‐nitropyridine) (DTNP) for deprotection and diselenide formation in protected selenocysteine‐containing peptides

In contrast to the large number of sidechain protecting groups available for cysteine derivatives in solid phase peptide synthesis, there is a striking paucity of analogous selenocysteine Se‐protecting groups in the literature. However, the growing interest in selenocysteine‐containing peptides and proteins requires a corresponding increase in availability of synthetic routes into these target molecules. It therefore becomes important to design new sidechain protection strategies for selenocysteine as well as multiple and novel deprotection chemistry for their removal. In this paper, we outline the synthesis of two new Fmoc selenocysteine derivatives [Fmoc‐Sec(Meb) and Fmoc‐Sec(Bzl)] to accompany the commercially available Fmoc‐Sec(Mob) derivative and incorporate them into two model peptides. Sec‐deprotection assays were carried out on these peptides using 2,2′‐dithiobis(5‐nitropyridine) (DTNP) conditions previously described by our group. The deprotective methodology was further evaluated as to its suitability towards mediating concurrent diselenide formation in oxytocin‐templated target peptides. Sec(Mob) and Sec(Meb) were found to be extremely labile to the DTNP conditions whether in the presence or absence of thioanisole, whereas Sec(Bzl) was robust to DTNP in the absence of thioanisole but quite labile in its presence. In multiple Sec‐containing model peptides, it was shown that bis‐Sec(Mob)‐containing systems spontaneously cyclize to the diselenide using 1 eq DTNP, whereas bis‐Sec(Meb) and Sec(Bzl) models required additional manipulation to induce cyclization. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Fmoc‐Sec(Xan)‐OH: synthesis and utility of Fmoc selenocysteine SPPS derivatives with acid‐labile sidechain protection

We report here the synthesis of the first selenocysteine SPPS derivatives which bear TFA‐labile sidechain protecting groups. New compounds Fmoc‐Sec(Xan)‐OH and Fmoc‐Sec(Trt)‐OH are presented as useful and practical alternatives to the traditional Fmoc‐Sec‐OH derivatives currently available to the peptide chemist. From a bis Fmoc‐protected selenocystine precursor, multiple avenues of diselenide reduction were attempted to determine the most effective method for subsequent attachment of the protecting group electrophiles. Our previously reported one‐pot reduction methodology was ultimately chosen as the optimal approach toward the synthesis of these novel building blocks, and both were easily obtained in high yield and purity. Fmoc‐Sec(Xan)‐OH was discovered to be bench‐stable for extended timeframes while the corresponding Fmoc‐Sec(Trt)‐OH derivative appeared to detritylate slowly when not stored at ?20 °C. Both Sec derivatives were incorporated into single‐ and multiple‐Sec‐containing test peptides in order to ascertain the peptides' deprotection behavior and final form upon TFA cleavage. Single‐Sec‐containing test peptides were always isolated as their corresponding diselenide dimers, while dual‐Sec‐containing peptide sequences were afforded exclusively as their intramolecular diselenides. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Differing views of the role of selenium in thioredoxin reductase

This review covers three different chemical explanations that could account for the requirement of selenium in the form of selenocysteine in the active site of mammalian thioredoxin reductase. These views are the following: (1) the traditional view of selenocysteine as a superior nucleophile relative to cysteine, (2) the superior leaving group ability of a selenol relative to a thiol due to its significantly lower pK _a and, (3) the superior ability of selenium to accept electrons (electrophilicity) relative to sulfur. We term these chemical explanations as the “chemico-enzymatic” function of selenium in an enzyme. We formally define the chemico-enzymatic function of selenium as its specific chemical property that allows a selenoenzyme to catalyze its individual reaction. However we, and others, question whether selenocysteine is chemically necessary to catalyze an enzymatic reaction since cysteine-homologs of selenocysteine-containing enzymes catalyze their specific enzymatic reactions with high catalytic efficiency. There must be a unique chemical reason for the presence of selenocysteine in enzymes that explains the biological pressure on the genome to maintain the complex selenocysteine-insertion machinery. We term this biological pressure the “chemico-biological” function of selenocysteine. We discuss evidence that this chemico-biological function is the ability of selenoenzymes to resist inactivation by irreversible oxidation. The way in which selenocysteine confers resistance to oxidation could be due to the superior ability of the oxidized form of selenocysteine (Sec-SeO₂ ⁻, seleninic acid) to be recycled back to its parent form (Sec-SeH, selenocysteine) in comparison to the same cycling of cysteine-sulfinic acid to cysteine (Cys-SO₂ ⁻ to Cys-SH).     

Sulphide detoxification in Hediste diversicolor and Marenzelleria viridis, two dominant polychaete worms within the shallow coastal waters of the southern Baltic Sea

The polychaete worms Marenzelleria viridis (Verrill 1873) and Hediste diversicolor (O.F. Müller) form the main part of the macro-zoobenthos in soft-bottomed shallow inlets of the Baltic Sea. Due to high eutrophication within these waters the animals are exposed to low oxygen and high sulphide concentrations. Specimens of both species from a low salinity location (S 8 ‰) were compared concerning their physiological abilities in coping with this hostile environment. Sulphide detoxification occurred in both polychaetes even during severe hypoxia with the main end-product being thiosulphate. In absence of sulphide nearly no end-products of anaerobic metabolism were found in the worms during moderate hypoxia (pO₂=7 kPa). In presence of hydrogen sulphide, succinate, a sensitive indicator of anaerobic metabolism, was accumulated in higher amounts at low sulphide concentrations (0.3 mM) already. Oxygen consumption and ATP production was determined in isolated mitochondria of both species. Both polychaetes were able to perform enzymatic sulphide oxidation in the mitochondria at concentrations up to 50 μM. This process was coupled with oxidative phosphorylation. At least in M. viridis sulphide respiration was not completely inhibited by cyanide, suggesting an alternative oxidation pathway, which by-passes the cytochrome-c-oxidase. The two species did not differ in the rate of sulphide detoxification, but H. diversicolor produced about as twice as much ATP from mitochondrial sulphide oxidation. Differences in mitochondrial sulphide oxidation are probably related to the different life strategies of the worms.     

Synthesis of peptides containing 5‐hydroxytryptophan,oxindolylalanine, N‐formylkynurenine and kynurenine

ROS, continuously produced in cells, can reversibly or irreversibly oxidize proteins, lipids, and DNA. At the protein level, cysteine, methionine, tryptophan, and tyrosine residues are particularly prone to oxidation. Here, we describe the solid phase synthesis of peptides containing four different oxidation products of tryptophan residues that can be formed by oxidation in proteins in vitro and in vivo: 5‐HTP, Oia, Kyn, and NFK. First, we synthesized Oia and NFK by selective oxidation of tryptophan and then protected the ${\bf \alpha}$ ‐amino group of both amino acids, and the commercially available 5‐HTP, with Fmoc‐succinimide. High yields of Fmoc‐Kyn were obtained by acid hydrolysis of Fmoc‐NFK. All four Fmoc derivatives were successfully incorporated, at high yields, into three different peptide sequences from skeletal muscle actin, creatin kinase (M‐type), and ${\bf \beta}$ ‐enolase. The correct structure of all modified peptides was confirmed by tandem mass spectrometry. Interestingly, isobaric peptides containing 5‐HTP and Oia were always well separated in an acetonitrile gradient with TFA as the ion‐pair reagent on a C₁₈‐phase. Such synthetic peptides should prove useful in future studies to distinguish isobaric oxidation products of tryptophan. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

The disulfide-coupled folding pathway of apamin as derived from diselenide-quenched analogs and intermediates.

The sequence of apamin, an 18 residue bee venom toxin, encloses all the information required for the correct disulfide-coupled folding into the cystine-stabilized alpha-helical motif. Three apamin analogs, each containing a pair of selenocysteine residues replacing the related cysteines, were synthesized to mimic the three possible apamin isomers with two crossed, parallel, or consecutive disulfides, respectively. Refolding experiments clearly revealed that the redox potential of selenocysteine prevails over the sequence encoded structural information for proper folding of apamin. Thus, selenocysteine can be used as a new device to generate productive and nonproductive folding intermediates of peptides and proteins. In fact, disulfides are selectively reduced in presence of the diselenide and the conformational features derived from these intermediates as well as from the three-dimensional (3D) structures of the selenocysteine-containing analogs with their nonnatural networks of diselenide/disulfide bridges allowed to gain further insight into the subtle driving forces for the correct folding of apamin that mainly derive from local conformational preferences.     

Fast cleavage of a diselenide induced by a platinum(II)-methionine complex and its biological implications

Platinum-based anticancer drugs such as cisplatin induce increased oxidative stress and oxidative damage of DNA and other cellular components, while selenium plays an important role in the antioxidant defense system. In this study, the interaction between a platinum(II) methionine (Met) complex [Pt(Met)Cl₂] and a diselenide compound selenocystine [(Sec)₂] was studied by electrospray ionization mass spectrometry, high performance liquid chromatography mass spectrometry, and ¹H NMR spectroscopy. The results demonstrate that the diselenide bond in (Sec)₂ can readily and quickly be cleaved by the platinum complex. Formation of the selenocysteine (Sec) bridged dinuclear complex [Pt₂(Met-S,N)₂(μ-Sec-Se,Cl)]³⁺ and Sec chelated species [Pt(Met-S,N)(Sec-Se,N)]²⁺ was identified at neutral and acidic media, which seems to result from the intermediate [Pt(Met-S,N)(Sec-Se)Cl]⁺. An accelerated formation of S-Se and S-S bonds was also observed when (Sec)₂ reacted with excessive glutathione in the presence of [Pt(Met)Cl₂]. These results imply that the mechanism of activity and toxicity of platinum drugs may be related to their fast reaction with seleno-containing biomolecules, and the chemoprotective property of selenium agents against cisplatin-induced toxicity could also be connected with such reactions.     

Heterotrimeric collagen peptides containing functional epitopes. Synthesis of single‐stranded collagen type I peptides related to the collagenase cleavage site

Synthetic collagen peptides containing larger numbers of Gly‐Pro‐Hyp repeats are difficult to purify by standard chromatographic procedures. Therefore, efficient strategies are required for the synthesis of higher molecular weight collagen‐type peptides. Applying the Fmoc/tBu chemistry, a comparative analysis of the standard stepwise chain elongation procedure on solid support with the procedure based on the use of the synthons Fmoc‐Gly‐Pro‐Hyp(tBu)‐OH and Fmoc‐Pro‐Hyp‐Gly‐OH was performed. The crude products resulting from the stepwise elongation procedure and from the use of Fmoc‐Gly‐Pro‐Hyp(tBu)‐OH clearly revealed large amounts of microheterogeneities that result from incomplete imino acid acylation as well as from diketopiperazine formation with cleavage of Gly‐Pro units from the growing peptide chain. Conversely, by the use of the Fmoc‐Pro‐Hyp‐Gly‐OH synthon, the quality of the crude products was significantly improved; moreover, protection of the Hyp side chain hydroxyl function is not required using the Fmoc/tBu strategy. With this optimized synthetic procedure, relatively large collagen‐type peptides were obtained in satisfactory yields as highly homogeneous compounds. Copyright © 1999 European Peptide Society and John Wiley & Sons, Ltd.     

Chemolithoutotrophic growth of Thiothrix ramosa

Thiothrix has been shown for the first time to be able to grow chemolithoautotrophically with thiosulphate or carbon disulphide as sole energy substrate. Thiosulphate served as the growth-limiting substrate for Thiothrix ramosa in chemostat culture. Maximum growth yield (Y_max) from yields at growth rates between 0.029–0.075 h^-1 was 4.0 g protein/mol thiosulphate oxidized. The key enzyme of the Calvin cycle, ribulose 1,5-bisphosphate carboxylase, was present in these cells, as were rhodanese, adenylyl sulphate (APS) reductase and sulphur-oxidizing enzyme. Thiosulphate-grown cells oxidized thiosulphate, sulphide, tetrathionate and carbon disulphide. Oxidation kinetics for sulphide, thiosulphate and tetrathionate were biphasic: oxygen consumption during the fast first phase of oxidation indicated oxidation of sulphide, and the sulphane moieties of thiosulphate and tetrathionate, to elemental sulphur, before further oxidation to sulphate. Kinetic constants for these four substrates were determined. T. ramosa also grew mixotrophically in batch culture on lactate with a number of organic sulphur compounds: carbon disulphide, methanethiol and diethyl sulphide. Substituted thiophenes were also used as sole substrates. The metabolic versatility of T. ramosa is thus much greater than previously realised.     

The synthesis and use of pp60-related peptides and phosphopeptides as substrates for enzymatic phosphorylation studies

A series of peptides and phosphopeptides corresponding to the auto-phosphorylation site of pp60^src, -Asn-Glu-Tyr⁴¹⁶-Thr-Ala-, were prepared by either Boc/solution or Fmoc/solid phase peptide synthesis and used as substrates to study their enzymatic phosphorylation by various casein kinases. The Tyr(P)-containing peptide, Asn-Glu-Tyr(P)-Thr-Ala, was prepared by the use of Fmoc-Tyr(PO₃Bzl₂)-OH in Fmoc/solid phase peptide synthesis followed by acidolytic treatment of the peptide-resin with 5% anisole/CF₃CO₂H. Both Asn-Glu-Tyr-Thr-Ala and Asn-Glu-Ser(P)-Thr-Ala were prepared by the Boc/solution phase peptide synthesis and employed hydrogenolytic deprotection of the protected peptides. Enzymatic phosphorylation studies established that (A) the Tyr residue acted as an unusual positive determinant for directing phosphorylation to the Thr-residue, (B) the rate of Thr-phosphorylation was markedly facilitated by a change from the Tyr-residue to the Tyr(P)-residue, and (C) a Ser(P)-residue was as effective as the Tyr(P)-residue in facilitating Thr-phosphorylation. A subsequent structure-function study using Asn-Glu-Phe-Thr-Ala, Asn-Glu-Tyr(Me)-Thr-Ala (prepared by Fmoc/solid phase peptide synthesis) and Asn-Glu-Cha-Thr-Ala (prepared by hydrogenation of Asn-Glu-Tyr-Thr-Ala) established that the rate of Thr-phosphorylation was influenced by the extent of hydrophobic-hydrophobic interactions by the aralkyl side-chain group (either aromatic or aliphatic) of the 416-residue with casein kinase-2; the rate of Thr-phosphorylation being decreased by the introduction of methyl or hydroxyl groups at the 4-position of the aromatic group {i.e. Tyr(Me) and Tyr respectively} but enhanced by the introduction of the hydrophilic phosphate group {i.e. as Tyr(P)}.     

Preventing aspartimide formation in Fmoc SPPS of Asp‐Gly containing peptides — practical aspects of new trialkylcarbinol based protecting groups

In our efforts to develop a universal solution to the problem of aspartimide formation in Fmoc SPPS, we investigated the application of our new β‐trialkylmethyl protected aspartic acid building blocks to the synthesis of peptides containing the Asp‐Gly motif. The N^α‐Fmoc aspartic acid β‐tri‐(ethyl/propyl/butyl)methyl esters were used in the synthesis of the classic model peptide scorpion toxin II (VKDGYI), and their effectiveness in minimising aspartimide formation during extended piperidine treatments was evaluated. Furthermore, we compared their efficacy against that of the commonly used approach of adding acids to the Fmoc deprotection solution. Finally, we applied our aspartic acid building blocks to the stepwise Fmoc SPPS of teduglutide, a human GLP‐2 analogue, whose synthesis is made challenging by extensive aspartimide formation. In all experiments, our approach led to almost complete reduction of aspartimide formation with accompanied suppression of aspartic acid epimerisation. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

Identification and Suppression of β-Elimination Byproducts Arising from the Use of Fmoc-Ser(PO3Bzl,H)-OH in Peptide Synthesis

The formation of 3-(1-piperidinyl)alanyl-containing peptides via phosphoryl β-elimination was identified from the application of Fmoc-Ser(PO₃Bzl,H)-OH in peptide synthesis as shown by RP-HPLC, ES-MS and ³¹P-NMR analysis. An N ^α -deprotection study using the model substrates, Fmoc-Xxx(PO₃Bzl,H)-Val-Glu(O^tBu)-Resin (Xxx = Ser, Thr or Tyr) demonstrated that piperidine-mediated phosphoryl β-elimination occurred in the N-terminal Ser(PO₃Bzl,H) residue at a ratio of 7% to the target phosphopeptide, and that this side reaction did not occur in the corresponding Thr(PO₃Bzl,H)- or Tyr(PO₃Bzl,H)- residues. The generation of 3-(1-piperidinyl)alanyl-peptides was also shown to be enhanced by the use of microwave radiation during Fmoc deprotection. An examination of alternative bases for the minimization of byproduct formation showed that cyclohexylamine, morpholine, piperazine and DBU gave complete suppression of β-elimination, with a 50% cyclohexylamine/DCM (v/v) deprotection protocol providing the crude peptide of highest purity. Piperidine-induced β-elimination was found only to occur in Ser(PO₃Bzl,H) residues that were in the N-terminal position, since the addition of the next residue in the sequence rendered the phosphoseryl residue stable to multiple piperidine treatments. The application of the alternative N ^α -deprotection protocol using 50% cyclohexylamine/DCM (v/v) is therefore recommended for deprotection of the Fmoc group from the Fmoc-Ser(PO₃Bzl,H) residue, with particular benefit anticipated for the synthesis of multiphosphoseryl peptides.     

Selenopeptide chemistry

This review focuses on the chemical aspects of the 21st proteinogenic amino acid, selenocysteine in peptides and proteins. It describes the physicochemical properties of selenium/sulfur and selenocysteine/cysteine based on comprehensive structural (X‐ray, NMR, CD) and biological data, and illustrates why selenocysteine is considered the most conservative substitution of cysteine. The main focus lies on the synthetic methods on selenocysteine incorporation into peptides and proteins, including an overview of the selenocysteine building block syntheses for Boc‐ and Fmoc‐SPPS. Selenocysteine‐mediated reactions such as native chemical ligation and dehydroalanine formation are addressed towards peptide conjugation. Selenopeptides have very interesting and distinct properties which lead to a diverse range of applications such as structural, functional and mechanistic probes, robust scaffolds, enzymatic reaction design, peptide conjugations and folding tools. Copyright © 2008 European Peptide Society and John Wiley & Sons, Ltd.     

Protected peptide p-nitroanilides by solid-phase synthesis

Peptide p-nitroanilides (peptide p> NAs) have found wide application as chromogenic substrates. An improved SPPS method to synthesize rapidly and in good yield a broad range of peptide pNAs under mild conditions will be presented here. To obtain a suitable carrier, the (4-aminophenyl)aminocarbonyloxy derivatives of Wang resin and Sasrin were synthesized. SPPS employing Fmoc/tBu-based protection followed by acidolytic cleavage yielded the (protected) peptide p-aminoanilide which was oxidized with sodium perborate in acetic acid to yield the(protected) peptide pNA. Side-chain protectionproved to be advantageous. Acid-labile peptide pNAs such as the proteasome substrate Z-Glu(OtBu)-Ala-Leu-pNA thus can be obtaineddirectly. The behavior of Cys, Met, Tyr and Trp being susceptible towards oxidation was studied more closely.     

Microwave-assisted solid-phase peptide synthesis of the 60–110 domain of human pleiotrophin on 2-chlorotrityl resin

A fast and efficient microwave-assisted solid phase peptide synthesis (MW-SPPS) of a 51mer peptide, the main heparin-binding site (60–110) of human pleiotrophin (hPTN), using 2-chlorotrityl chloride resin (CLTR-Cl) following the 9-fluorenylmethyloxycarbonyl/tert-butyl (Fmoc/tBu) methodology and with the standard N,N′-diisopropylcarbodiimide/1-hydroxybenzotriazole (DIC/HOBt) coupling reagents, is described. An MW-SPPS protocol was for the first time successfully applied to the acid labile CLTR-Cl for the faster synthesis of long peptides (51mer peptide) and with an enhanced purity in comparison to conventional SPPS protocols. The synthesis of such long peptides is not trivial and it is generally achieved by recombinant techniques. The desired linear peptide was obtained in only 30 h of total processing time and in 51% crude yield, in which 60% was the purified product obtained with 99.4% purity. The synthesized peptide was purified by reversed phase high performance liquid chromatography (RP-HPLC) and identified by electrospray ionization mass spectrometry (ESI-MS). Then, the regioselective formation of the two disulfide bridges of hPTN 60–110 was successfully achieved by a two-step procedure, involving an oxidative folding step in dimethylsulfoxide (DMSO) to form the Cys⁷⁷–Cys¹⁰⁹ bond, followed by iodine oxidation to form the Cys⁶⁷–Cys⁹⁹ bond.     

Investigation of peptide thioester formation via N→Se acyl transfer

Native chemical ligation is widely used for the convergent synthesis of proteins. The peptide thioesters required for this process can be challenging to produce, particularly when using Fmoc‐based solid‐phase peptide synthesis. We have previously reported a route to peptide thioesters, following Fmoc solid‐phase peptide synthesis, via an N→S acyl shift that is initiated by the presence of a C‐terminal cysteine residue, under mildly acidic conditions. Under typical reaction conditions, we occasionally observed significant thioester hydrolysis as a consequence of long reaction times (~48 h) and sought to accelerate the reaction. Here, we present a faster route to peptide thioesters, by replacing the C‐terminal cysteine residue with selenocysteine and initiating thioester formation via an N→Se acyl shift. This modification allows thioester formation to take place at lower temperatures and on shorter time scales. We also demonstrate how application of this strategy also accelerates peptide cyclization, when a linear precursor is furnished with an N‐terminal cysteine and C‐terminal selenocysteine. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Synthesis and biological characterization of a series of analogues of ω-conotoxin gvia

The 27-residue polypeptide ω-conotoxin GVIA (ω-CgTx), from the venom of the cone shell Conus geographus, blocks N-type neuronal calcium channels. It contains three disulphide bridges. We reporte here the synthesis and biological characterization of a seires of analogues in which one disulphide has been replaced by substitution of appropriate Cys residues with Ser, viz. [Ser^1,16]-ω-CgTx, [Ser^8,19]-ω-CgTx, [Ser^15,26]-ω-CgTx, [Ser¹⁶]-ω-CgTx_8-27 and [Ser¹⁵]-ω-CgTx_1-19. All syntheses were conducted manually using either Boc or Fmoc methodology. Deprotected peptides were oxidized to their bridged forms using either aerial oxidation or aqueous dimethyl sulphoxide. Peptides were purified using RP-HPLC, and their purity and identity were checked by RP-HPLC, capillary electrophoresis and mass spectrometry. Inhibition of neuronal N-type calcium channels was assessed as the inhibition of the twitch responses of rat vas deferens stimualted with single electrical pulses at 20 second intervals. None of these analogues was biologically active, suggesting that the disulphides play an important role in maintaining biological activity.     

Solid-phase synthesis of tyrosyl H-phosphonopeptides and methylphosphonopeptides

Phosphopeptides are a useful tool for the investigation of phosphorylation as a reversible post-translational modification. There is a growing interest in using mimics of phosphoamino acids involved in phosphorylation in order to study the enzymes concerned in these processes. These mimics should contain a non-hydrolysable or isoelectrically modified phosphate moiety to be used as a specific inhibitor of phosphatases and kinases. We introduce solid-phase synthesis of H- and methylphosphonopeptides as a new class of mimics of phosphotyrosyl peptides. The peptides were synthesized on solid phase using the standard fluorenyl-methyloxycarbonyl (Fmoc) strategy. Tyrosine residues were incorporated as allyl-protected derivatives, which were selectively deprotected on the resin by treatment with Pd(PPh₃)₄. The peptide resin carrying the side-chain unprotected tyrosine of the model peptide Gly-Gly-Tyr-Ala was phosphonylated with di-tert-butyl-N,N-diethyl-phosphoramidite in the presence of ¹H-tetrazole, yielding H-phosphonopeptides after trifluoroacetic acid (TFA) cleavage. Alternatively, phosphonylation of the unprotected tyrosine with O-tert-butyl-N,N-diethyl-P-methylphosphonamidite catalysed by ¹H-tetrazole and followed by oxidation led to the methylphosphonopeptides after TFA cleavage. We obtained both the H-phosphonopeptides and the methylphosphonopeptides of the tetrapeptide in high yields and purities above 90%, according to reversed-phase high-performance liquid chromatography (RP-HPLC). To investigate the general applicability of our new methodology, we synthesized phosphonopeptides up to 13 amino acids long, corresponding to recognition sequences of tyrosine kinases. After cleavage and deprotection, all phosphonopeptides were obtained in high yields and purities of about 90%, as shown by mass spectrometry. The only by-product found was the unmodified peptide. © 1997 European Peptide Society and John Wiley & Sons, Ltd.     

Effects of phenolic substances on metabolism of exogenous indole-3-acetic acid in maize stems

Four-day-old stem segments of Zea mays L. cv. Seneca 60 were treated sequentially with phenolic substances and indole-3-acetic [2-¹⁴C] acid ([2-¹⁴C]IAA). Formation of bound IAA was rapid, but a pretreatment with p-coumaric acid, ferulic acid or 4-methylumbelliferone decreased the level of bound IAA. The decrease is not likely related to the effect of the phenolics on enzymic oxidation of IAA, since the level of free IAA was not limiting and the activity of ferulic acid in enzymic oxidation of IAA is different from that of p-coumaric acid and 4-methylum-belliferone. Apparently these compounds inhibited the formation of bound IAA and consequently caused an accumulation of free IAA. In contrast, caffeic acid, protocatechuic acid and 2,3-dihydro-2, 2-dimethyl-7-benzofuranol had little effect. After the uptake of IAA there was a slow but steady incorporation of the radioactivity into the 80% ethanol-insoluble, 1 M NaOH-soluble fraction. Phenolic substances also affected this process. The compounds which are cofactors of IAA-oxidase increased the incorporation while those which are inhibitors of IAA-oxidase decreased the incorporation. Results suggest that the phenolics also affected the enzymic oxidation of IAA in vivo in the same way as in vitro.     

Diphenyl Diselenide-Induced Seizures in Rat Pups: Possible Interaction with Glutamatergic System

The aims of the present study were to investigate the possible involvement of glutamatergic system in seizures induced by diphenyl diselenide in rat pups (postnatal day, 12-14) and to evaluate the role of oxidative stress in seizures induced by diphenyl diselenide/glutamate. Glutamate (4 g/kg of body weight) administered in association with diphenyl diselenide (500 mg/kg of body weight) increased the latency for the appearance of the first seizure episode, reduced lipid peroxidation levels and catalase, Na⁺,K⁺-ATPase and δ-ALA-D activities. At the lowest dose (5 mg/kg of body weight), diphenyl diselenide reduced the appearance of seizure episodes induced by glutamate but did not alter the latency for the onset of the first episode. Glutamate uptake was inhibited in glutamate, diphenyl diselenide (the highest dose) and in the association of diphenyl diselenide (both doses) and glutamate groups. Pre-treatment with a N-methyl-D-aspartate (NMDA) receptor antagonist, MK-801 (5S,10R-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate), significantly prolonged the latency for the onset for the first convulsive episode. A non-NMDA receptor antagonist, DNQX (6,7-dinitroquinoxaline-2,3-dione), did not protect seizures induced by diphenyl diselenide. The results of the present study demonstrated that: (a) when diphenyl diselenide and glutamate were administered concomitantly in pups, glutamate was the main responsible for the neurotoxic effects; (b) oxidative stress was not involved in glutamate-induced seizures; (c) NMDA glutamatergic receptors, were at least in part, involved in diphenyl diselenide- induced seizures; and (d) diphenyl diselenide, at the lowest dose, protected seizures induced by glutamate.