Synthesis of dihydroquinopimaric acid conjugates with amino acids

Synthesis of dihydroquinopimaric acid amides and their 2β-succinyl and 2β-phthalyl derivatives containing residues of amino acids was carried out for the first time. Antiviral properties of the compounds synthesized were investigated.     

Synthesis and biological activity of amino acid conjugates of abscisic acid

We prepared 19 amino acid conjugates of the plant hormone abscisic acid (ABA) and investigated their biological activity, enzymatic hydrolysis by a recombinant Arabidopsis amidohydrolases GST-ILR1 and GST-IAR3, and metabolic fate in rice seedlings. Different sets of ABA-amino acids induced ABA-like responses in different plants. Some ABA-amino acids, including some that were active in bioassays, were hydrolyzed by recombinant Arabidopsis GST-IAR3, although GST-ILR1 did not show hydrolysis activity for any of the ABA-amino acids. ABA-L-Ala, which was active in all the bioassays, an Arabidopsis seed germination, spinach seed germination, and rice seedling elongation assays, except in a lettuce seed germination assay and was hydrolyzed by GST-IAR3, was hydrolyzed to free ABA in rice seedlings. These findings suggest that some plant amidohydrolases hydrolyze some ABA-amino acid conjugates. Because our study indicates the possibility that different plants have hydrolyzing activity toward different ABA-amino acids, an ABA-amino acid may function as a species-selective pro-hormone of ABA.     

Xanthine oxidase catalyzes the oxidation of retinol

In mammals, xanthine oxidase (E.C. 1.17.3.2) catalyzes the hydroxylation of a wide variety of heterocyclic substrates such as purines, pyrimidines, and pterins, in addition to aldehydes [1] as all-trans-retinaldehyde [2-5]. Here, we show that buttermilk xanthine oxidase was capable to oxidizing all-trans-retinol (t-ROL) to all-trans-retinaldehyde (t-RAL) that was successively oxidized to all-trans-retinoic acid (t-RA). A rise in the enzyme activity, when t-ROL-CRBP complex was assayed, with respect to the free t-ROL, was observed. Furthermore, treatment of the enzyme with Na2S and glutathione resulted in a significant increment in catalytic activity toward t-ROL and t-RAL, due to the reconstitution of the native structural organization of the molybdenum centre of molybdopterin cofactor of the desulfo form of xanthine oxidase.     

Effect of lunularic acid analogues on liverwort growth and IAA oxidation

The growth inhibitory activity of lunularic acid and a number of analogues has been examined in liverwort gemmaling and cress root growth tests. Lunularic acid was no more active than a wide range of similar compounds and no clear correlation between structure and activity was observed. The effects of these compounds, and of liverwort extracts, on in vitro IAA-oxidase activity was also examined.     

Synthesis of amino acid conjugates of 1,1′-dimethylferrocene: New chiral conjugates

Syntheses of diastereomeric mixtures of 1,1′-dimethylferrocene-2-Ala-OMe (3) and 1,1′-dimethylferrocene-3-Ala-OMe (4) are reported by peptide coupling of L-Ala-OMe to the enantiomeric mixtures of the corresponding acids, 1,1′-dimethylferrocene-2-carboxylic acid (1) and 1,1′-dimethylferrocene-3-carboxylic acid (2).     

Diamine oxidase from millet catalyzes the oxidation of 1, 3-diaminopropane

Diamine oxidase was purified sixty-fold from millet shoots. The partially purified enzyme of 150 kDa oxidized 1, 3-diaminopropane (1, 3-DAP) to 3-aminopropionaldehyde. The K_m values were 9.1×10⁻⁵M for 1, 3-DAP and 6.3×10⁻⁴M for putrescine. Extracts of shoots of prosomillet, maize and barley also contained an activity that oxidized 1, 3-DAP.     

Anti-HIV activity of a series of cosalane amino acid conjugates

The binding of the anti-HIV agent cosalane to CD4 is thought to involve ionic interactions of negatively charged carboxylates of the ligand with positively charged residues on the surface of the protein. The purpose of the present study was to examine the hypothesis that the two carboxyl groups of cosalane could be sacrificed through conjugation to amino acids, and the anti-HIV activity still be retained, provided that at least two new carboxyl groups are contributed by the amino acid residues.     

Synthesis and cytotoxic activities of beta-carboline amino acid ester conjugates

Beta-carboline represents a class of compounds with potent anti-tumor activity by intercalating with DNA. To further enhance the cytotoxic potency and bioavailability of beta-carboline, a series of novel beta-carboline amino acid ester conjugates were designed and synthesized, and the cytotoxic activities of these compounds were tested using a panel of human tumor cell lines. In addition, the membrane permeability of these compounds was evaluated in vitro using a Caco-2 cell monolayer model. The beta-carboline amino acid ester conjugates demonstrated improved cytotoxic activity compared to the parental beta-carbolines. In particular, the Lys/Arg conjugates were the most potent analogs with an IC(50) value of 4 and 1 microM against human cervical carcinoma cells. The low interaction energy of Arg conjugate based on molecular modeling may contribute to its enhanced cytotoxicity. Taken together, this study provided new insights into structure-activity relationships in the beta-carboline amino acid ester conjugates and identified the beta-carboline Lys/Arg conjugates as promising lead compounds for further in vivo biological and molecular evaluation.     

Long chain acyl CoA synthetase 4 catalyzes the first step in peroxisomal indole-3-butyric acid to IAA conversion

Indole-3-butyric acid (IBA) is an endogenous storage auxin important for maintaining appropriate indole-3-acetic acid (IAA) levels, thereby influencingprimary root elongation and lateral root development. IBA is metabolized into free IAA in peroxisomes in a multistep process similar to fatty acid β-oxidation. We identified LONG CHAIN ACYL-COA SYNTHETASE 4 (LACS4) in a screen for enhanced IBA resistance in primary root elongation in Arabidopsis thaliana. LACSs activate substrates by catalyzing the addition of CoA, the necessary first step for fatty acids to participate in β-oxidation or other metabolic pathways. Here, we describe the novel role of LACS4 in hormone metabolism and postulate that LACS4 catalyzes the addition of CoA onto IBA, the first step in its β-oxidation. lacs4 is resistant to the effects of IBA in primary root elongation and dark-grown hypocotyl elongation, and has reduced lateral root density. lacs6 also is resistant to IBA, although both lacs4 and lacs6 remain sensitive to IAA in primary root elongation, demonstrating that auxin responses are intact. LACS4 has in vitro enzymatic activity on IBA, but not IAA or IAA conjugates, and disruption of LACS4 activity reduces the amount of IBA-derived IAA in planta. We conclude that, in addition to activity on fatty acids, LACS4 and LACS6 also catalyze the addition of CoA onto IBA, the first step in IBA metabolism and a necessary step in generating IBA-derived IAA.

An enhancer mutant revealed an acyl-CoA synthetase that catalyzes CoA addition to indole-3-butryic acid, required for the β-oxidation steps necessary to generate indole-3-butryic acid-derived IAA.     

Aromatic amino acid oxidation products as antioxidants

The accumulation of UV photolysis products of amino acids tyrosine and tryptophan, which possess antioxidant activity, has been studied by the method of luminol-dependent chemiluminescence. The amount of antioxidant products was judged by the value of the total antioxidant potential of a UV-irradiated solution, the measure of which was the distance between the peaks of the chemiluminescence curve in the system 2,2′-azo-bis(2-amidinopropane) hydrochloride + luminol with a UV-irradiated and an unirradiated sample (induction period, τ _i ). Simultaneously, the absorption and fluorescence spectra of unirradiared and UV-irradiated amino acid solutions were recorded. It was shown that exposure of a tryptophan solution to radiation led to accumulation of a fluorescent product N-formyl kynurenine (λ_em = 325 nm, λ_max = 440 nm), and the curve of its accumulation was similar to the growth of antioxidant potential. When a tyrosine solution was irradiated, the main fluorescent product was dityrosine (λ_em = 310 nm, λ_max = 415 nm). Nevertheless, the dose dependences of the formation of dityrosine and the total antioxidant potential were completely different. It was found that another product of tyrosine UV photolysis, dihydroxyphenylalanine, possessed pronounced antioxidant activity. It was concluded that the main antioxidant produced under UV irradiation of tryptophan is formyl kynurenine, and under irradiation of tyrosine it is dihydroxyphenylalanine.     

Antioxidants as aromatic amino acid oxidation products

The accumulation of UV photolysis products of amino acids tyrosine and tryptophan, which possess an antioxidant activity, has been studied by the method of luminol-activated chemiluminescence. The amount of antioxidant products was judged by the value of the total antioxidant potential of a UV-irradiated solution, the measure of which was the distance between the peaks of the chemiluminescence curve in the system 2,2'-azo-bis(2-amidinopropane)hydrochloride + luminol in a UV-irradiated and an unirradiated samples (induction period, tau(i)). Simultaneously, the absorption and fluorescence spectra of unirradiared and UV-irradiated amino acid solutions were recorded. It was shown that, upon the exposure of a tryptophan solution to radiation, the accumulation of the fluorescent product N-formyl kynurenine (lambda(em) = 325 nm, lambda(max) = 440 nm) occures, and the curve of its accumulation was similar to the curve of growth of tau(i) photoproducts produced during UV-radiation. When a tyrosine solution was irradiated, the main fluorescent product was dityrosine (lambda(em) = 310 nm, lambda(max) = 415 nm). Nevertheless, the dose dependencies of the formation of dityrosine, and the total antioxidant potential (tau(i)) were completely different. It was found that another product of tyrosine UV-photolysis, dioxyphenylalanine, possessed a pronounced antioxidant activity. It was concluded that the main antioxidants produced under UV-irradiation of tryptophan is formyl kynurenine, and under the irradiation of tyrosine, dioxyphenylalanine.     

Phytochelatin synthase catalyzes key step in turnover of glutathione conjugates

Conjugation of xenobiotic compounds and endogenous metabolites to glutathione is an ubiquitous process in eukaryotes. In animals, the first and rate-limiting step of glutathione-S-conjugate metabolism is characterized by the removal of the aminoterminal glutamic acid residue of glutathione. In plants, however, glutathione-S-conjugates are generally metabolized by removal of the carboxylterminal glycine residue of the tripeptide glutathione to give rise to the S-glutamylcysteinyl-derivative. Purification of the glutathione-conjugate catabolizing activity from cell suspension cultures of the plant Silene cucubalus indicated that phytochelatin synthase catalyzes the first step of the pathway. Heterologously expressed phytochelatin synthase from Arabidopsis efficiently converted S-bima ne-glutathione to S-bimane-glutamylcysteine, the formation of which was unequivocally identified by mass spectrometry. No further products, such as S-derivatives of phytochelatins, were observed. Several different glutathione-S-conjugates served as substrates for the enzyme and were processed to the corresponding glutamylcysteinyl-adducts. Affinity-purified phytochelatin synthase preparations required divalent heavy metal ions such as Cd(2+), Zn(2+) or Cu(2+) for detectable turnover of glutathione-S-conjugates. Characterization of the enzymatic properties of phytochelatin synthase argues for both cellular functions of the gamma-glutamylcysteinyl-dipeptidyltransferase: (1) formation of heavy-metal binding peptides and (2) degradation of glutathione-S-conjugates. Mechanistically, the former role is the result of gamma-glutamylcysteinyl transpeptidation onto glutathione or derivatives thereof, while the catabolic function reflects transpeptidation of S-glutamylcysteinyl-adducts onto the acceptor molecule water. Thus, phytochelatin synthase seems to fulfil a second crucial role in glutathione metabolism.     

Preparation of amino acid conjugates of betulinic acid with activity against human melanoma.

Betulinic acid has been coupled with a series of amino acids at C-28 carboxylic acid position and the toxicity of the derivatives has been evaluated against cultured human melanoma (MEL-2) and human epidermoid carcinoma of the mouth (KB) cell lines. A number of amino acid conjugates of betulinic acid showed improved water solubility as well as selective cytotoxicity. This investigation demonstrates that amino acid conjugates of betulinic acid can produce potentially important derivatives, which may be developed as antitumor agents.     

Effects of IAA and four IAA conjugates on morphogenesis and callus growth from tomato leaf discs

The effects of indole-3-acetic acid (IAA) and four IAA conjugates, indoleacetylalanine (IAAla), indoleacetylaspartic acid (IAAsp), indoleacetylglycine (IAGly), and indoleacetylphenylalanine (IAPhe), on growth and morphogenesis in tomato leaf discs in vitro were examined. Free IAA stimulated root initiation in the absence of cytokinin and stimulated callus growth in the presence of 0.89 M benzylaminopurine (BAP). Free IAA also inhibited shoot initiation obtained with 8.9 M BAP. The activities of the IAA conjugates depended on the conjugating amino acid, the concentration of the conjugate, and the response being measured. IAAsp had little or no activity in promoting root initiation or callus growth or in inhibiting shoots, while IAPhe was similarly inactive except at the highest concentration tested (100 M). IAAla and IAGly were both very active in inhibiting shoots and promoting callus growth, but were much less active in stimulating rooting, except at 100 M, at which concentration they were as effective as free IAA. Thin-layer chromatography of the IAA conjugates revealed that IAAla, IAGly and IAPhe were largely stable to autoclaving, but that IAAsp underwent some hydrolysis to products identical with free IAA and aspartic acid. Pretreatment of seedlings with IAA, IAAla or IAGly altered the subsequent shoot initiation response from leaf discs on media with and without IAA.     

Chemiluminescence associated with amino acid oxidation mediated by hypochlorous acid

Although most amino acids readily react with hypochlorous acid (HOCl), only the reaction involving tryptophan (Trp) produces a measurable chemiluminescence (CL). Most of this luminescence takes place after total consumption of HOCl when the process is carried out in an excess of Trp. The quantum yield of the process is relatively low (2 × 10^?8 Einstein/mol HOCl reacted). The luminescence is attributed to free radical‐mediated secondary reactions of the initially produced chloramines. This is supported by experiments showing that the chloramines produced when HOCl reacts with alanine are able to induce Trp chemiluminescence, and that this luminescence is partially quenched by free radical scavengers. The spectral changes and the effect of pH upon the observed luminescence are compatible with light emission from products produced in the free radical oxidation of Trp. Copyright © 2002 John Wiley & Sons, Ltd.     

Perturbing the DNA sequence selectivity of metallointercalator-peptide conjugates by single amino acid modification.

Metallointercalator-peptide conjugates that provide small molecular mimics to explore peptide-nucleic acid recognition have been prepared. Specifically, a family of peptide conjugates of [Rh(phi)(2)(phen')](3+) [where phi = 9,10-phenanthrenequinone diimine and phen' = 5-(amidoglutaryl)-1,10-phenanthroline] has been synthesized and their DNA-binding characteristics examined. Single amino acid modifications were made from the parent metallointercalator-peptide conjugate [Rh(phi)(2)(phen')](3+)-AANVAIAAWERAA-CONH(2), which targets 5'-CCA-3' site-specifically. Moving the glutamate at position 10 in the sequence of the appended peptide to position 6 {[Rh(phi)(2)(phen')](3+)-AANVAEAAWARAA-CONH(2)} changed the sequence preference of the metallointercalator-peptide conjugate to 5'-ACA-3'. Subsequent mutation of the glutamate at position 6 to arginine {[Rh(phi)(2)(phen')](3+)-AANVARAAWARAA-CONH(2)} caused more complex changes in DNA recognition. Thermodynamic dissociation constants were determined for these metallointercalator-peptide conjugates by photoactivated DNA cleavage assays with the rhodium intercalators. At 55 degrees C in the presence of 5 mM MnCl(2), [Rh(phi)(2)(phen')](3+)-AANVAIAAWERAA-CONH(2) binds to a 5'-CCA-3' site with K(d) = 5.7 x 10(-)(8) M, whereas [Rh(phi)(2)(phen')](3+)-AANVAEAAWARAA-CONH(2) binds to its target 5'-ACA-3' site with K(d) = 9.9 x 10(-8) M. The dissociation constant for [Rh(phi)(2)(phen')](3+) with random-sequence DNA is 7.0 x 10(-7) M. Structural models have been developed and refined to account for the observed sequence specificities. As with much larger DNA-binding proteins, with these metal-peptide conjugate mimics, single amino acid changes can lead to single or multiple base changes in the DNA site targeted.     

Indirect oxidation of amino acid phenylhydrazides by mushroom tyrosinase

We have investigated oxidation of amino acid phenylhydrazides by mushroom tyrosinase in the presence of 4-tert-butylcatechol and N-acetyl-L-tyrosine. Spectrophotometric measurements showed gradual disappearance of 4-tert-butyl-o-benzoquinone, generated by oxidation of 4-tert-butylcatechol with sodium periodate, after addition of amino acid phenylhydrazides. However, the presence of the phenylhydrazides did not influence the concentration of 4-tert-butyl-o-benzoquinone formed during enzymatic oxidation. Oxygen consumption measurements demonstrated that in a mixture both compounds were oxidized but the reaction rate was proportional to the concentration of the catechol. In the oxidation of N-acetyl-L-tyrosine addition of phenylhydrazides shortened the lag period, indicating that they acted as reducing agents, converting N-acetyl-L-dopaquinone to N-acetyl-L-dopa. In HPLC analysis of the oxidation 4-tert-butylcatechol and the phenylhydrazide of Boc-tryptophan only the N-protected amino acid and 4-tert-butyl-o-benzoquinone were detected as final products. In the presence of the natural substrates the oxidation of amino acid phenylhydrazides required much smaller amounts of the enzyme and was up to 40 times faster than the reaction carried out without these compounds. These results demonstrate that tyrosinase can oxidize phenylhydrazides indirectly through o-quinones. This reaction explains the inhibitory effect of agaritine, a natural amino acid hydrazide, on melanin formation and the inhibitory effects of other hydrazine derivatives on tyrosinase described in the literature.     

Free radical-mediated oxidation of free amino acids and amino acid residues in proteins

Summary. We summarize here results of studies designed to elucidate basic mechanisms of reactive oxygen (ROS)-mediated oxidation of proteins and free amino acids. These studies have shown that oxidation of proteins can lead to hydroxylation of aromatic groups and aliphatic amino acid side chains, nitration of aromatic amino acid residues, nitrosylation of sulfhydryl groups, sulfoxidation of methionine residues, chlorination of aromatic groups and primary amino groups, and to conversion of some amino acid residues to carbonyl derivatives. Oxidation can lead also to cleavage of the polypeptide chain and to formation of cross-linked protein aggregates. Furthermore, functional groups of proteins can react with oxidation products of polyunsaturated fatty acids and with carbohydrate derivatives (glycation/glycoxidation) to produce inactive derivatives. Highly specific methods have been developed for the detection and assay of the various kinds of protein modifications. Because the generation of carbonyl derivatives occurs by many different mechanisms, the level of carbonyl groups in proteins is widely used as a marker of oxidative protein damage. The level of oxidized proteins increases with aging and in a number of age-related diseases. However, the accumulation of oxidized protein is a complex function of the rates of ROS formation, antioxidant levels, and the ability to proteolytically eliminate oxidized forms of proteins. Thus, the accumulation of oxidized proteins is also dependent upon genetic factors and individual life styles. It is noteworthy that surface-exposed methionine and cysteine residues of proteins are particularly sensitive to oxidation by almost all forms of ROS; however, unlike other kinds of oxidation the oxidation of these sulfur-containing amino acid residues is reversible. It is thus evident that the cyclic oxidation and reduction of the sulfur-containing amino acids may serve as an important antioxidant mechanism, and also that these reversible oxidations may provide an important mechanism for the regulation of some enzyme functions.