Ureido Group Research Articles

ChemInform Abstract: Intramolecular Ureido and Amide Group Participation in Reactions of Carbonate Diesters.

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Stimulation of Bovine Brain Phospholipase C Activity by Myelin Basic Protein Requires Arginyl Residues in Peptide Linkage

We reported previously a highly purified phosphatidylinositol-specific phospholipase C (PI-PLC) from bovine brain and from human myelin which was stimulated by myelin basic protein. In this paper we report that the stimulation of the PI-PLC activity by myelin basic protein (MBP) requires arginine residues in peptide linkage. MBP and poly-L-arginine were able to stimulate the PI-PLC activity by 250% while other basic poly amino acids were unable to stimulate the PI-PLC activity. Neither free arginine nor benzoyl-arginine ethylester was able to stimulate the activity of the enzyme. These results suggested a requirement for the guanidino group of arginine and arginine in peptidyl linkage. The arginyl residues of MBP were modified chemically with 1,2-cyclo-hexanedione, or enzymatically by cholera toxin which ADP-ribosylated arginyl groups, or by peptidylarginine deiminase which converted the guanidino group of arginine to the ureido group of citrulline. ADP-ribosylation did not affect the stimulation while the 1,2-cyclohexane-dione modified MBP and the peptidylarginine deiminasetreated MBP showed a reduced ability to stimulate the PI-PLC activity which correlated with the number of arginyl residues modified. Sequence analysis of the peptidylarginine deiminase-treated MBP established that specific arginyl residues had been converted to citrulline to a greater extent than others. When 70% of Arg 25 and Arg 31 were converted to citrulline little stimulatory activity remained, whereas the conversion of 100% of Arg 170 did not affect the ability of C1 to stimulate the enzyme. A role for "active" arginine in this MBP peptide is suggested by our data.

Interference of Carbohydrates in the Quantitation of Protein-Bound Citrulline by Amino Acid Analysis

After cation-exchange chromatographic separation of the charge isomers of human myelin basic protein, the citrulline-containing component was purified from the unbound fraction by gel permeation chromatography. Sephadex G75 (superfine) resolved high- and low-molecular-weight contaminants from the 18.5K myelin basic protein. However, carbohydrates leached from the column material by the acidic eluant interfered with citrulline quantitation by amino acid analysis. It appears highly probable that during acid hydrolysis of the protein, glucose reacts with citrulline, the ureido group of the latter forming a condensation product with the sugar which subsequently undergoes further chemical degradation to products which are not easily identifiable. Methods for removing the interfering sugars prior to amino acid analysis are discussed.

Intramolecular ureido and amide group participation in reactions of carbonate diesters

The cyclization of ethyl and phenyl 2-ureidophenylcarbonates in H 2 O at 30 o C involves two discrete steps with benzoxazolinone as the final product. The formation of benzoxazolinone is quantitative. Phenol is released in the initial step from both esters in an apparent OH - -catalyzed reaction, which shows that intramolecular nucleophilic attack by the ureido group is via an anionic species. The pH-rate constant profile for the second step in the reaction of the ethyl ester is sigmoidal K app =8.9

15N NMR, 1H NOE difference spectroscopy and conformation of N‐(o‐aminophenyl)‐N′‐alkylureas

AbstractThe 15N NMR spectra of five N‐(o‐aminophenyl)‐N′ substituted ureas and their hydrochloride salts were recorded in DMSO. N‐1 was more shielded in these ureas than in N‐phenyl‐N′‐methylurea. The shifts of N‐3 varied with the substituents. Proton exchanges at N‐3 were observed when the spectra of the hydrochloride salts were recorded. The preferential trans‐trans orientation of the protons with respect to the carbonyl in the ureido group was confirmed by NOE difference spectroscopy. In the solid, the conformation of N(o‐aminophenyl)‐N′‐methylurea, which was determined by x‐ray diffraction, was found to be similar to the suggested conformation in solution; the 15N shifts of this compound in the solid state were determined by 15N cross‐polarization magic angle spinning NMR.

M-[ 125I]iodoaniline: a useful reagent for radiolabeling biotin

Biotinyl- m-[ 125I]iodoanilide (BIA) was synthesized by coupling biotin to m-[ 125I]iodoaniline via a mixed anhydride reaction. m-[ 125I]Iodoaniline was produced from the tin precursor, which was prepared using a palladium catalyzed reaction of hexabutylditin with m-bromoaniline. The radioiodinated BIA derivative is characterized by a stable amide and/or intact ureido group on the biotin molecule; it may thus be a useful carrier for targeting radionuclides to avidin-conjugated antibodies previously localized on tumors.

Reactivity of two anti-galactosyl ceramide antibodies towards myelin basic protein

Two anti-galactosyl ceramide antibodies (polyclonal Ab142 and a monoclonal antibody) were characterized in terms of their reactivity towards purified lipids and myelin basic protein. Polyclonal Ab142 is a rabbit anti-mouse galactosyl ceramide (Gal C) IgG. Antigenic recognition is dependent on both galactose and ceramide since neither could inhibit galactosyl ceramide binding by more than 10%. MAb-Gal C is a monoclonal antibody raised in mice against galactosyl ceramide. Binding of MAb-Gal C to Gal-C was equally inhibited by ceramide and galactose to approximately 50%, indicating that both groups are important for antibody recognition. MAb-Gal C was also shown to be reactive with the structurally related lipids, sphingomyelin and sulfatide. Polyclonal Ab142, although raised against Gal C, was shown to be 3-fold more reactive with component 8 (C-8) of myelin basic protein than Gal C. On the other hand, the MAb-Gal C which also reactive with C-8 than with Gal C. Neither of these antibodies were reactive with component 1 (C-1) of myelin basic protein. An anti-MBP IgG was shown to be reactive with C-1 and C-8 but unreactive with Gal C. In competitive inhibition ELISA, C-8 was able to compete out 44% and 41% of Gal C binding to polyclonal Ab142 and MAb, respectively. The reverse competition demonstrated that Gal C could inhibit 75% of C-8 binding to both antibodies. D-galactose was unable to inhibit C-8 binding to either antibody, whereas ceramide was as efficient as Gal C. When the anti-citrulline antibody was used, it reacted well with C-8 both in solution and in lipid vesicles. Some reaction was obtained against Gal C in solution but not when Gal C was present in lipid vesicles. We propose that the reactivity of anti-Gal C antibodies towards C-8 of myelin basic protein is related to similarities between the ceramide backbone of Gal C and the ureido group of the citrullinyl residue in C-8.

The biosynthesis of xanthocillin-X monomethyl ether in dichotomomyces cejpii. Experiments on the origin of the isocyanide carbon atoms

Precursor feeding experiments establish that the isocyanide groups in xanthocillin-X monomethyl ether (XME)1 do not originate from (i) compounds associated with C1-tetrahydrofolate metabolism; (ii) the ureido group of citrulline, carbamoyl phosphate, or potassium cyanate; or (iii) cyanide and arguably related compounds. However the results with D-[U-13C] glucose show that the origin of the isocyanide groups is metabolically closer to glucose than is the origin of the backbone; DL-[1 -13C] glyceraldehyde and D-[1-13C]glucose are not specific precursors.

Conformational Preferences of Modified Nucleic Acid Bases N6-Methyl-N6-(N-Threonylcarbonyl) Adenine and 2-Methylthio-N6-(N-Threonylcarbonyl) Adenine by the Quantum Chemical PCILO Calculations

Conformational preferences of the hypermodified nucleic acid bases N6-methyl-N6-(N-threonylcarbonyl) Adenine, m6tc6 Ade, and 2-methylthio-N6-(N-threonylcarbonyl) Adenine, mS2 tc6 Ade, have been studied theoretically using the quantum chemical PCILO (Perturbative Configuration Interaction using Localized Orbitals) method. The multidimensional conformational space has been searched using selected grid points formed by combining the various torsion angles which take the favoured values obtained from energy variation with respect to each torsion angle individually. In m6 tc6 Ade and mS 2tc6 Ade alike the threonylcarbonyl substituent preferably orients away (distal) from the imidazole moiety of the adenine ring. And as in the simpler N6-(N-threonylcarbonyl) Adenine, tc6 Ade, the atoms in the ureido group as well as the amino acid carbon atoms C(12) and C(13) remain coplanar with the purine base. As in tc6 Ade, this conformation is stabilized by the intramolecular hydrogen bond between N(11)H of the amino acid and N(1) of the adenine base. The N6-methyl protons, in m6 tc6 Ade, take trans-staggered orientation with respect to the C(6)-N(6) bond. The preferred orientation of the 2-methylthio group is cis to the C(2)-N(3) bond in mS 2tc6 Ade. This is in marked contrast to the modified nucleic acid base 2-methylthio-N6-(delta 2-isopentenyl) Adenine, mS 2i6 Ade, where the 2-methylthio group orients trans to the C(2)-N(3) bond, causing a change in the preferred orientation of the isopentenyl component on methylthiolation. The present results thus indicate that unlike in the isopentenyl adenine the role of further chemical substitutions in threonylcarbonyl adenine may be indirect and less pronounced.

Intramolecular nucleophilic attack by urea nitrogen. Reactivity–selectivity relationships for the general acid–base catalysed cyclisations of ureido acids and esters

The cyclisation of N-substituted hydantoic acids involves general acid–base catalysed attack of urea nitrogen on the CO2H group. The variation of the Brønsted exponents for the reactions of the N-methyl and N-phenyl derivatives (2) and (3) allows a choice between kinetically equivalent mechanisms. Stereoelectronic effects on the nucleophilic reactions of urea NH are considered, and a mechanism involving an initial rotation about the terminal C–N bond of the ureido group is suggested.

Biochemical characterization of a new highly cardioselective beta-adrenoceptor antagonist.

P0160 (1-phenyl-3-(2-(3-(2-cyanophenoxy)-2-hydroxypropyl)amino) ethylhydantoin HCl) is an aryloxypropanolamine which contains a ureido group as part of the hydantoin ring. This molecule was synthesized to obtain a more cardioselective beta-adrenoceptor blocker. Preliminary data have shown that it is as potent as propranolol and four times more cardioselective than atenolol in pharmacological tests in-vitro and in the conscious rat. In the present study we evaluated the interaction of P0160 with beta-adrenoceptors by radioreceptor binding studies and by measuring adenylate cyclase activity coupled to beta-adrenoceptors. The data indicate that P0160 binds with nanomolar affinity to beta-adrenoceptors labelled with [3H]DHA in the rat heart, but with micromolar affinity in the rat lung. Its binding is stereospecific, the S-(-)isomer being 200 times more active than the R-(+) form. P0160's selectivity between cardiac beta 1- and beta 2-receptors was 1388, about 60 times that for metoprolol. Analysis of the thermodynamic characteristics of P0160's interaction with rat heart beta-adrenoceptors indicated antagonist properties of the same order of magnitude as propranolol, as confirmed by adenylate cyclase studies. These data indicate that P0160 is a potent, specific and selective beta 1-adrenoceptor antagonist, and give a molecular explanation for the cardioselective activity found in pharmacological tests.

Sequence of the conversion of pyrimido [5,4-e]-1,2,4-triazinediones to imidazo[4,5-e]-1,2,4-triazinones

The conversion of pyrimido[5,4-e]-1,2,4-triazinediones to imidazo[4,5-e]-1,2,4-triazinones is a multistage process including the hydrolysis of the C(5)-N(6) amide bond, the closure of the ureido group to the imidazolidinone ring, decarboxylation, acid oxidation.

Synthesis, structure, and hydrolysis of esters of strained and unstrained N-phosphonylureas

Compounds 1–5 were prepared to compare reactivity patterns of cyclic and acyclic phosphonylurea esters. The rates and products of reactions of phosphonylurea esters (1–3) with hydroxide in aqueous acetonitrile were analyzed. In these compounds the phosphonate moiety is in a strained five-membered ring, which also contains the ureido group. Structural determination of 1 by X-ray crystallography indicates that the five-membered ring is planar and the internal ring angle at phosphorus is 93.1°. The endocyclic N—C—N angle of the ureido group is 111°. The compounds undergo hydrolysis in alkaline aqueous acetonitrile at 35 °C with a rate about 106 times that of analogues (4, 5) in which the phosphonate group is exocyclic to the ureido ring. Compound 1 undergoes alkaline hydrolysis (k = 9.0 × 103 M−1 s−1) to release the phenoxy group to give 6. The hydrolysis of alkyl esters 2 (k = 2.4 × 104 M−1 s−1)and 3(k = 1.3 × 103 M−1 s−1) leads to cleavage of the endocyclic P—N bond, producing 7 and 8 respectively. The exocyclic alkyl esters (4 and 5) also cleave at the P—N bond with respective rate constants of 6.5 × 10−3 M−1 s−1 and 4.4 × 10−2 M−1 s−1. The data are consistent with a mechanism in which hydroxide adds to 1 to form a pentacoordinate phosphorus intermediate with the phenoxy group in an equatorial position and the ureido ring in apical and equatorial positions (with nitrogen apical). The departure of the urea group is slower than pseudorotation of the intermediate and expulsion of phenoxide. In the isomerized intermediate, phenoxy is apical but the methylene group of the ring, which has low apicophilicity, must also be apical. Reactions of 2 and 3, which have more basic oxygen leaving groups, occur with P—N cleavage because expulsion from the isomerized intermediate in those cases is not sufficiently fast. These results fit reaction patterns at phosphorus that are determined by ring strain and electronegativity of ligands. Contributions from effects due to antiperiplanar interactions between bonding and nonbonding electrons are not detected.

Biosynthesis of the spermidine and guanidino units in the glycocinnamoylspermidine antibiotic cinodine.

The biosynthesis of the spermidine and guanidino groups has been studied with carbon-14 and carbon-13 labeled intermediates. Arginine, citrulline and ornithine are incorporated in good efficiency. The guanidino group of arginine and the ureido group of citrulline both label the guanidino group on the hexose sugar. None of the ureido groups in the antibiotic was enriched. It is likely that citrulline is converted to arginine before use in the biosynthesis. Arginine, citrulline and ornithine are incorporated as the four carbon unit of spermidine. All of the labeled 5 carbon from ornithine or from citrulline appears adjacent to the secondary amine in the four carbon unit of spermidine. This would indicate that unbound putrescine is not an immediate precursor of spermidine.

N-Carbamoyl-DL-aspartic acid

CsHaN205, PL a = 6.438 (2), b = 7.486 (3), c = 8.048 (4)A, a = 72.2(1), fl = 80.8(1), y = 76.4 (1) °, D m = 1.65 (1) (flotation), D c = 1.64 Mg m -3, Z = 2. Final R = 0.095 for 1205 observed reflections. The molecule assumes the sterically least favourable conformation with the side chain carboxyl group staggered between the a-carboxyl group and the N atom attached to C '~. The ureido group takes part in two specific interactions involving two nearly parallel hydrogen bonds in one and two convergent hydrogen bonds in the other.

Enzymic conversion of agmatine to putrescine in Lathyrus sativus seedlings. Purification and properties of a multifunctional enzyme (putrescine synthase).

The participation of a multifunctional enzyme (a single polypeptide with multiple catalytic activities (14)) has been demonstrated in the conversion of agmatine to putrescine in Lathyrus sativus seedlings. This enzyme (putrescine synthase) with inherent activities of agmatine iminohydrolase, putrescine transcarbamylase, ornithine transcarbamylase, and carbamate kinase has been purified to homogeneity and has Mr = 55,000. In the presence of inorganic phosphate, the enzyme catalyzed the stoichiometric conversion of agmatine and ornithine to putrescine and citrulline, respectively. The different activities associated with the enzyme copurified with near constancy in their specific activity. The enzyme catalyzed phosphorolysis and arsenolysis of N-carbamyl putrescine. The multifunctionality of putrescine synthase was also supported by 1) activity staining, 2) intact transfer of the ureido-14C group from labeled NJ-carbamyl putrescine to ornithine to form citrulline, and 3) the affinity of the enzyme toward structurally and functionally related affinity matrices. An agmatine cycle is proposed wherein N-carbamyl putrescine arising from the agmatine iminohydrolase reaction is converted to putrescine and citrulline, with the ureido group of N-carbamyl putrescine being transferred intact to ornithine. Preliminary results indicate that this series of reactions is also present in other plants.

Biochemical Aspects of Urea Cycle Disorders

In this paper I will review the biochemistry of the urea cycle of mammalian liver and point out the important clinical implications of this biochemistry. Second, I will consider some problems that arise in the use of human liver enzyme assays for evaluating deficiencies of urea cycle enzymes. Finally, I will comment on our own research concerning enzyme induction of the urea cycle of mammalian liver. In Fig 1 the urea cycle is reviewed. Within the mitochondrion of the liver cell, ammonium cation combines with bicarbonate anion and 2 moles of adenosine 5'-triphosphate (ATP) in the presence of an activator, N-acetylglutamate, to form carbamylphosphate. Enzyme 1, which carries out this reaction is carbamylphosphate synthetase I (CPS1). There is also another enzyme, CPS2, in the cytoplasm of liver cells about which more will be said later. Once carbamylphosphate is formed, the carbamyl group is transferred to the δ-amino group of ornithine to form citrulline. The enzyme in this reaction, enzyme 2, is ornithine transcarbamylase (OTC). Citrulline escapes, by a process other than active transport, into the cytoplasm where it is conjugated with aspartate to form argininosuccinate; this is accomplished by enzyme 3, argininosuccinate synthetase (AS). Argininosuccinate, a double amino acid, is then cleaved to yield fumarate, and the nitrogen of aspartate moves to arginine. The enzyme in this reaction, enzyme 4, is argininosuccinate lyase (AL); its trivial name is argininosuccinase. Finally, arginine undergoes cleavage of its ureido group by enzyme 5, arginase, to form urea and regenerate ornithine. Ornithine is then taken up into the mitochondria by an active transport process to combine again with carbamylphosphate.

The coordinating properties of d-biotin.

The coenzyme d-biotin offers in its anionic form to metal ions 3 possible binding sites: the carboxylate group of the valerate side chain, the ureido residue of the 2-imidazolidone ring, and the thioether sulfur of the tetrahydrothiophene ring; the coordinating properties of these groups are summarized and compared. Hydrogen bond formation of the ureido group has also been observed, and hydrogen bonding may possibly be important in biotin-bicarbonate recognition. The aliphatic part of the valeric acid side chain can undergo hydrophobic interactions. Such interactions and/or the stereoselective sulfur-metal ion coordination could be the means for a correct 'fixation' of the biotinyl moiety at the surface of a protein, thus creating the active enzyme-substrate complex.

Participation in the formation of iodo ureas from 3-buten-1-ol derivatives: A reinvestigation

Treatment of O-substituted derivatives of 3-buten-1-ol with silver cyanate and iodine, followed by ammonia, gave cyclic derivatives 5 and 6. These compounds were previously assigned the linear structures 3 and 4, respectively. Evidence in support of the proposed structural reassignment was obtained from high-field 1H and 13C NMR studies. Both 5 and 6 undergo loss of the ureido group upon treatment with water to give 9 and 10, and 12, respectively. Potential mechanisms for the observed reactions are discussed.

Catabolism of L-arginine by Pseudomonas aeruginosa.

Pseudomonas aeruginosa is known to break down arginine by the arginine deiminase pathway. An additional pathway has now been found whereby arginine is converted to putrescine with agmatine and N-carbamoylputrescine as intermediates. The following enzyme activities belonging to this pathway were detected in crude extracts: arginine decarboxylase (EC 4.1.1.19), which catalyses the release of CO2 from arginine to give agmatine; agmatine deiminase (EC 3.5.3.12), which degrades agmatine to N-carbamoylputrescine; and N-carbamoylputrescine amidinohydrolase (EC 3.5.3.-), which then removes the ureido group of carbamoylputrescine. In crude extracts, arginine decarboxylase activity was stimulated by pyridoxal phosphate, Mg2+ and by the products of the catabolic pathway, putrescine and spermidine. Growth of P. aeruginosa on arginine as the sole carbon and nitrogen source markedly increased the activity of arginine decarboxylase. Agmatine and N-carbamoylputrescine induced the synthesis of agmatine deiminase and N-carbamoylputrescine hydrolase. Addition of succinate or citrate to medium containing arginine or agmatine led to repression of the enzymes involved in the arginine decarboxylase pathway. Moreover, the repression of agmatine deiminase and N-carbamoylputrescine hydrolase was further increased when P. aeruginosa was grown in media with agmatine plus glutamine or agmatine plus succinate and ammonia. This suggests that the expression of the agmatine pathway may be regulated by carbon catabolite repression as well as nitrogen catabolite repression.