Carboxylic Acid Side Chain Research Articles

Prosthetic group content and ligand binding properties of a spinach catalase

A recently discovered form of spinach catalase that contains both a novel heme and protoheme as prosthetic groups has been characterized using immunological and spectroscopic techniques. The enzyme appears to be a dimer of identical M r 60 000 monomers. Extraction of the non-covalently bound prosthetic groups, followed by thin-layer chromatography of the extract, suggested that the novel heme contains four carboxylic acid side-chain groups. The resonance Raman spectrum of the resting enzyme indicates that the protoheme prosthetic group is five-coordinate and high-spin. The enzyme was shown to bind formate, azide and cyanide. Cyanide and azide binding to catalase are biphasic, suggesting the existence of two different binding sites for cyanide and azide in the enzyme. Results obtained from EPR and resonance Raman spectroscopies also support the hypothesis that two different ligand-binding sites are present in the enzyme. Western blots suggest that the M r 60 000 peptide of the novel heme-containing catalase is similar or identical to that of a previously characterized, exclusively protoheme-containing, tetrameric catalase.

Potentiometric selectivity study of crown ethers containing four ring oxygen atoms and benzoxymethyl or carboxylic acid side chains as ionophores for lithium and potassium

AbstractFive crown ethers with four ring oxygen atoms and different types of pendant groups were used to prepare ion‐selective electrodes by coating a membrane containing the crown ether on the tip of a silver wire incorporated in a flow cell. A flow injection analysis study was conducted to determine the potentiometric selectivities of the crown ethers for lithium, sodium, potassium, magnesium, and calcium ions. It was found that crown ethers with neutral side arms show selectivities for lithium over other ions, while those of acidic side arms show selectivities for potassium over the other ions. The effect of the addition of trioctylphosphine oxide to the electrode matrix was also investigated. A comparison is made between the potentiometric selectivities of the crown ethers for certain metal ions and their selectivities in extracting the ions into an organic phase.

Spectroscopic Properties of Siroheme Extracted from Sulfite Reductases

Siroheme has been extracted from sulfite reductases and its properties in aqueous solution have been investigated by optical absorption, electron paramagnetic resonance (EPR), and magnetic circular dichroism (MDC) spectroscopy. The absorption spectrum of siroheme exhibits a marked pH dependence, and two p K values, 4.2 and 9.0, were determined by pH titration in the range 2–12. The first p K (4.2) is thought to correspond to the ionization of the carboxylic acid side-chains on the tetrapyrrole rings, and the second p K (9.0) is attributed to displacement of the axial ligand chloride by hydroxide. The binding of the strong field ligands, CO, NO, and cyanide, were investigated by UV-visible absorption and, in the case of the cyanide complex, by low-temperature EPR and MCD spectroscopies. CO and NO were able to reduce and bind to siroheme without additional reducing agent. The EPR spectrum of the isolated siroheme (chloride-ferrisiroheme) exhibits an axial signal with g XXX = 6.0 and g= 2.0, typical of high-spin ferric hemes (S = 5 2 ), whereas the cyanide-complexed siroheme exhibits an approximately axial signal with g XXX = 2.38 and g = 1.76 that is indicative of a low-spin ferric heme (S = 1 2 ). The low-temperature MCD spectra and magnetization data for the as-isolated and cyanide-complexed ferrisiroheme are entirely consistent with the interpretation of the EPR spectra. The results for ferrosiroheme indicate that the siroheme remains high spin (S = 2) and low spin (S = 0) on reduction of the as-isolated and cyanide-complexed siroheme, respectively. The isolated siroheme expressed sulfite reductase activity but the assessable catalytic cycle was much less than that of the native enzyme, showing the importance of the protein environment.

In vitro mutated beta subunits from the F1-ATPase of the thermophilic bacterium, PS3, containing glutamine in place of glutamic acid in positions 190 or 201 assembles with the alpha and gamma subunits to produce inactive complexes.

Using site-directed mutagenesis, Glu-190 or Glu-201 of the beta subunit of the F1-ATPase from the thermophilic bacterium PS3 were replaced with glutamine. It was possible to reconstitute complexes of the mutated beta subunits with alpha and gamma subunits, but the complexes did not have ATPase activity. It is concluded that carboxylic acid side chains of Glu-190 and Glu-201 of the beta subunit are essential for catalytic activity of F1-ATPase.

Synthesis of C‐3 side‐chain carboxylic acid cannabinoid derivatives

AbstractBased upon the structural similarities of cannabinoid derivatives which inhibit the biosynthesis of prostaglandins with those of the potent inhibitor, indomethacin, several cannabinoid derivatives possessing C‐3 side chain carboxylic acid functionalities were prepared.

ChemInform Abstract: A NOVEL SYNTHESIS OF (.+‐.)‐CARBACYCLIN

AbstractWie im Form‐elschema angedeutet, wird aus dem Cyclohexenderivat (IIc) über ozonolytische Ringöffnung zum Dialdehyd (III) der Hydroxycarbaldehyd (IV) hergestellt.

Degradation and Epimerization Kinetics of Moxalactam in Aqueous Solution

The kinetics of epimerization and degradation of moxalactam in aqueous solution was investigated by HPLC. The pH-rate profiles of the degradation and epimerization were determined separately over the pH range of 1.0–11.5 at 37°C and constant ionic strength 0.5. The degradation and simultaneous epimerization were followed by measuring both of the residual R- and S-epimers of moxalactam and were found to follow pseudo-first-order kinetics. The degradation was subjected to hydrogen ion and hydroxide ion catalyses and influenced by the dissociation of the side chain phenolic group. The epimerization rates were influenced significantly in the acidic region by the dissociation of the side chain carboxylic acid group and in the basic region by hydroxide ion catalysis. The pH-degradation rate profile of moxalactam showed a minimum degradation rate constant between pH 4.0 and 6.0. The pH-epimerization rate profiles of moxalactam showed minimum epimerization rate constants at pH 7.0. The epimerization rate constants of the R- and S-epimers were not very different.

A novel synthesis of (.+-.)-carbacyclin.

Carbacyclin (2, R=H) was synthesized by the application of a new method for introducing the carboxylic acid side chain via dehydrative decarboxylation of the β-hydroxy carboxylic acid (19).

15N-Coprobiliverdin, a New Model Chromophore

The synthesis of 15N-labeled coprobiliverdin III, a bile pigment with four conjugated pyrrole nuclei and four carboxylic acid side chains, is described. Coprobiliverdin is structurally characterized by chemical and various spectroscopic methods (e.g. chromic acid degradation, UV-vis-, IR-, mass spectroscopy). The results of 15N NMR studies reveal the potential value of this compound as a probe for structural interactions in biliproteins and as a reference for the future investigation of natural bile pigments deriving from coproheme

Urobiliverdin, a New Bile Pigment Deriving from Uroporphyrin

5-Aminolevulinic acid is incubated with a crude enzyme extract from Rhodopseudomonas spheroides, mutant R 26. The formed porphyrins (main product: uroporphyrin III) are isolated. Incorporation of iron, ring-splitting by coupled oxydation and subsequent iron removal leads to a mixture of pigments, from which urobiliverdin, a new bile pigment with eight carboxylic acid side chains, is isolated. It is characterized by its chromatographic behaviour, chromic acid degra­dation and UV-vis spectroscopy.

The role of conjugation reactions in enhancing biliary secretion of bile acids.

Shortening the five-carbon carboxylic acid side chain of cholic acid by one methylene group gave rise to a bile acid (norcholate) that was not a substrate for the bile acid-conjugating enzymes. The metabolism and biliary secretion of norcholate in intact liver was examined in the isolated perfused rat liver system. When rat livers were perfused with 14-20 microM solutions of norcholate for 10 min, norcholate was found in the unconjugated form in liver, venous effluent and bile. Neither tauronorcholate nor glyconorcholate was detectable by high-pressure liquid chromatography or fast-atom-bombardment mass spectrometry. The kinetics of hepatic uptake and biliary secretion of norcholate was compared with that for cholate, taurocholate and chemically synthesized tauronorcholate. The latter three bile acids were completely cleared from the perfusate and efficiently secreted into the bile. However, norcholate was incompletely extracted from the perfusate, and this was shown to be at least partially due to its relatively lower rate of hepatic uptake. Furthermore, the rate of norcholate secretion into bile was greatly reduced relative to the secretion of cholate or chemically synthesized tauronorcholate, even though the concentration of norcholate in the liver was comparatively high. These data demonstrate that the conjugation of bile acids greatly facilitates their secretion into bile.

Synthesis and characterization of coprobiliverdin III, a new model chromophore

The synthesis of coprobiliverdin III, a new bile pigment with four conjugated pyrrole nuclei and four carboxylic acid side chains, is described. Coprobiliverdin is structurally characterized by chromic acid degradation, mass spectroscopy, UV vis and NMR spectroscopy.

Blue color, metal content, and substrate binding in 4-hydroxyphenylpyruvate dioxygenase from Pseudomonas sp. strain P. J. 874.

Purified preparations of 4-hydroxyphenylpyruvate dioxygenase from Pseudomonas sp. strain P. J. 874 are blue, epsilon 595-850 approximately 2.6 +/- 0.5 (n = 6) mM-1 cm-1. Iron and zinc were the only metals detected by x-ray fluorescence of an enzyme preparation and the mean content in different preparation as determined by atomic absorption spectroscopy was determined by atomic absorption spectroscopy was 0.95 +/- 0.17 (n = 6) and 0.68 +/- 0.27 (n = 7) mol/mol 150-kilodalton tetramer, respectively. It is yet unclear if zinc is a contaminant or may be given a structural role. Results with iron chelators and reductants showed that the 595-nm absorbance is linked to enzyme-bound Fe3+ and that reduction of iron, which occurs concomitantly with disappearance of the color, is required for enzyme activity. The enol tautomer of 4-hydroxyphenylpyruvate appeared to form 2:1 a complex with enzyme-bound Fe3+, which may be the cause of the long known substrate inhibition of the enzyme. Iron chelation also seemed to be involved in the inhibition by other substrate analogues, i.e. substituted catechols and those with one phenolic hydroxyl group in ortho position to short carboxylic acid side chains. Together, substrate analogue, pH, and modification studies indicated that the tautomerizable keto group with a double bond in 3-4 position favors productive substrate binding to Fe2+ and a base with a pK alpha of approximately 6.4.

Inhibition of protohaem ferro-lyase by N-substituted porphyrins. Structural requirements for the inhibitory effect.

N-Methyl mesoporphyrin was a powerful inhibitor of protohaem ferro-lyase in vitro, whereas N-ethyl mesoporphyrin and N-methyl coproporphyrin were not and neither was the newly described green pigment produced by giving rats ethylene. This suggests that the size of the substituent at a pyrrole nitrogen and also the number of carboxylic acid side chains of the substituted porphyrin are important for the inhibitory effect. Evidence that N-methyl mesoporphyrin inhibited the enzyme, whereas the ethylene-derived pigment did not, was also obtained in vivo.

Chain fission of poly-L-glutamic acid by a water-soluble carbodiimide

Summary Water-soluble carbodiimides are used extensively to modify carboxylic acid side chains in proteins. There have been no reports of side reactions involving the peptide backbone. This study reports the extensive cleavage of the peptide backbone in poly-L-glutamic acid by the water-soluble carbodiimide N-ethyl-N′-(γ-dimethylaminopropyl) carbodiimide in aqueous solution at pH 5.0 and room temperature. Some of the conditions and limitations of this reaction are examined. A scheme for the reaction mechanism, involving spatially neighboring carboxylic acid groups, is proposed.

Tryptic cleavage of a peptide at modified aspartic acid

Conversion of the carboxylic acid side chain of aspartic acid in the peptide pyroGlu·Asp·Phe·amide to a carboxamidomethylamine side chain (structure l ) results in the tryptic hydrolysis of the peptide at the Asp-Phe bond.

PH Dependence of magnesium ion binding to prothrombin fragment 1 and γ-carboxyglutamic acid-containing peptides via 25MG NMR

The binding of magnesium ions to two tripeptides, L -Arg- D -Gla- D -Gla-OMe and Z- L -Arg(NO 2)- D -Gla- D -Gla-OMe, and to bovine prothrombin fragment 1 as a function of pH has been monitored by 25Mg NMR spectroscopy. Binding to the tripeptide was dependent on peptide ionizations occurring at pH 4.6 – 4.8. The pH dependence of magnesium ion binding to fragment 1 reveals two inflection points 4.2 may be attributed to the deprotonation of the third side chain carboxylic acid group of the double γ-carboxyglutamic acid sequence. The origin of the increased binding of magnesium ions to fragment 1 at pH values above 7 is unknown.

Microbiological oxidation of the pentyl side chain of cannabinoids.

Syncephalastrum racemosum ATCC 18192 and Mycobacterium rhodochrous ATCC 19067 partially degrade the n-pentyl side chain of cannabidiol, cannabinol, delta8-tetrahydrocannabinol and delta9-tetrahydrocannabinol. Carboxylic acid and alcohol side chain derivatives are major metabolites.

Potential antitumor agents. 27. Quantitative structure-antileukemic (L1210) activity relationships for the .omega.[4-(9-acridinylamino)phenyl]alkanoic acids

Simple carboxylic acid derivatives of 9-anilinoacridine (e.g., 1,R=-COOH) provide high experimental antileukemic (L1210) activity. The homologous 1'-(CH2)nCOOH congeners also prove active, and there is a parabolic interrelationship between maximum increase in life span in L1210 tests and Rm values used as a measure of agent lipophilic--hydrophilic balance. The corresponding carboxamides [1'-(CH2)nCONH2] provide a similar parabolic relationship, which has an optimum Rm value displaced from that of the acids. Earlier examined 1'-NHSO2(CH2)nCH3 variants, the 3-NHCOCH3 congeners of these, and the carboxamide [1'-(CH2)nCONH2] and sulfonamide [1'-(CH2)nSO2NH2] homologues can be treated as one group and a correlation equation derived that is identical with that for the carboxamide variants alone. The optimum Rm value for this group is displaced from that of the acids by the equivalent of 1.8 log P units on the octanol--water scale. In this drug series a carboxylic acid residue acts as an acceptable hydrophilic unit, providing a log P contribution intermediate between that of the un-ionized acid and the totally ionized carboxylate anion. Quantitative effects of acridine ring substituents on L1210 activity differ in analogues containing either carboxylic acid or alkanesulfonanilde side chains, supporting the view that different site-binding orientations may be involved with these two drug classes.

Detection of the stable addition of carbodiimide to proteins

A simple convenient synthesis is described for radioactive 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride. This reagent makes possible convenient detection of the extent of internal rearrangement and the stable addition of carbodiimide to protein when the compound is used in chemical modification studies. A survey of the reaction of carbodiimide alone with eleven typical proteins indicates that the reagent itself may be suitable for selective modification of only a few residues, including carboxylic acid side chains partially buried on the surface or in active sites. This would provide a powerful and selective tool for probing residue function in enzymes.