Improvement of hyaluronic acid enzymatic stability by the grafting of amino-acids

Injection of hyaluronic acid (HA)-based hydrogels has proven to provide many therapeutic benefits. To increase the stability of HA-based products against enzymatic digestion, we modified hyaluronic acid by grafting various amino acids on its carboxylic group and then evaluated the enzymatic stability of the various conjugates in presence of a hyaluronidase. Our results showed that all amino acid-modified HA polymers were more resistant to degradation compared to the native HA albeit with variation according to the amino acids. Amino acids with carboxylate groups such as aspartic acid or with hydroxyl functions (threonine, serine or tyrosine) conferred a particularly strong resistance to HA towards enzymatic digestion. The HA-amino acid products were then cross-linked with butanediol diglycidyl ether (BDDE). The swelling properties of the formed hydrogels appeared close to native HA whereas the increased resistance towards hyaluronidase digestion remained. These results suggest that amino acid-modified HA derivatives can become promising material for viscosupplementation or drug delivery.     

Intermediates of the gamma-glutamyl cycle in mouse tissues. Influence of administration of amino acids on pyrrolidone carboxylate and gamma-glutamyl amino acids.

GAMMA-Glutamyl transpeptidase, gamma-glutamyl cyclotransferase, L-pyrrolidone carboxylate hydrolase, gamma-glutamylcysteine synthetase and glutathione synthetase, the enzymes of the gamma-glutamyl cycle, were found in mouse brain, liver and kidney. The activity of L-pyrrolidone carboxylate hydrolase was many times lower than the activities of the other enzymes, and thus the conversion of L-pyrrolidone carboxylate to L-glutamate is likely to be the rate-limiting step of the cycle. The specificity of gamma-glutamyl cyclotransferase from mouse tissues was similar to that from rat tissues. The concentration of pyrrolidone carboxylate and gamma-glutamyl amino acids, intermediates of the gamma-glutamyl cycle, was determined by a gas chromatographic procedure coupled with electron capture detection. Administration of L-2-aminobutyrate, an amino acid that is utilized as substrate in the reaction catalyzed by gamma-glutamylcysteine synthetase, led to a large accumulation of gamma-glutamyl-2-aminobutyrate and pyrrolidone carboxylate in mouse tissues. L-Methionine-RS-sulfoximine, an inhibitor of gamma-glutamylcysteine synthetase, abolished the increase in concentration of pyrrolidone carboxylate. No accumulation of pyrrolidone carboxylate was observed after L-cysteine. The separate administration of several protein amino acids had little effect on the concentration of pyrrolidone carboxylate; however formation of small amounts of the corresponding gamma-glutamyl derivatives (e.g. gamma-glutamylmethionine and gamma-glutamylphenylalanine) was detected. These intermediates are probably formed by transpeptidation between glutathione and the corresponding amino acid, catalyzed by gamma-glutamyl transpeptidase. The concentration of pyrrolidone carboxylate increased significantly after administration of a mixture containing all protein amino acids, the highest increase occurring in the kidney. The results suggest that two separate pathways for the formation of gamma-glutamyl amino acids and pyrrolidone carboxylate exist in vivo. One of these results from the function of gamma-glutamylcysteine synthetase in glutathione synthesis. The other pathway involves the amino-acid-dependent degradation of glutathione, mediatedby gamma-glutamyl transpeptidase. Only very small amounts of free intermediates are apparently derived from the latter pathway, suggesting that the gamma-glutamyl amino acids formed in this pathway are either enzyme-bound or are directly hydrolyzed to glutamate and free amino acid.     

Functionalized cobalt clusters. Properties of carboxylate clusters (CO)6Co2HCCCOOH and (CO)10Co4HCCCOOH as related to their structures

Reaction of propiolic acid (HCCCOOH) with dicobalt octacarbonyl (Co₂(CO)₈) and tetracobalt dodecacarbonyl (Co₄(CO)₁₂) leads to the organometallic carboxylic acids (CO)₆Co₂HCCCOOH (1) and (CO)₁₀Co₄HCCCOOH (2) in good yield. Both organometallic compounds show a cobalt carbonyl core bonded to a carboxylate function. The structure of the acetylene-carboxylic group in both clusters deviates from that of ethylene. The C(1)C(2)C(3) fragment is half way between acrylic and acetylene-carboxylic acid. The comparative acidity of the carboxylic group measured in methanol reveals that (1) is a stronger acid than (2), but less acidic than propiolic acid. Both organometallic carboxylic acids are thermally decomposed into phases with high metal content at relatively low temperatures. Fenske-Hall calculations on this series of cobaltocarbonyl cluster carboxylic acids confirm that the Co(CO)₃ donates electron density to the HCCCOOH fragment, thus decreasing the acidity of the carboxylic function. The redox properties and the electronic spectra are also well correlated to the HOMO-LUMO gap thus calculated by this non-parametric SCF method.     

Production and recovery of propionic and acetic acids in electrodialysis culture of Propionibacterium shermanii

The simultaneous production and recovery of propionic and acetic acids in cultures of Propionibacterium shermanii were examined while maintaining the concentrations of these acids in the fermentation broth at low values with electrodialysis to alleviate end product inhibition. By this method, maximum cell concentration increased 1.36 fold and the average productivities of propionic and acetic acids increased 1.4 fold and 1.31 fold, respectively, compared to cultures without electrodialysis. The mass ratio of propionic and acetic acids recovered was the same as that for the acids produced by fermentation. The inhibitory effect of propionic acid was more significant on the growth of cells than on the production of propionic acid. Some kinetic equations are presented for simulating the dry cell concentration in the fermentor and the concentrations of propionic and acetic acids in the permeate solution of an electrodialysis culture.     

Recovery of lactic acid from fermentation broth by the two-stage process of nanofiltration and water-splitting electrodialysis

A two-stage process of nanofiltration and water-splitting electrodialysis was investigated for lactic acid recovery from fermentation broth. In this process, sodium lactate is isolated from fermentation broth in the first stage of nanofiltration by using an NTR-729HF membrane, and then is converted to lactic acid in the second stage by water-splitting electrodialysis. To determine the optimal operating conditions for nanofiltration, the effects of pressure, lactate concentration, pH and known added impurities were studied. Lactate rejection was less than 5%, magnesium rejection approximated 45%, and calcium rejection was at 40%. In subsequent water-splitting electrodialysis, both the sodium lactate conversion to lactic acid and sodium hydroxide recovery, were about 95%, with a power requirement of 0.9∼1.0 kWh per kg of lactate.     

Understanding substrate misrecognition of hydrogen peroxide dependent cytochrome P450 from Bacillus subtilis

Cytochrome P450_BSβ, a H₂O₂-dependent cytochrome P450 catalyzing the hydroxylation of long-alkyl-chain fatty acids, lacks the general acid–base residue around the heme, which is indispensable for the efficient generation of the active species using H₂O₂. On the basis of the crystal structure of the palmitic acid bound form of cytochrome P450_BSβ, it was suggested that the role of the general acid–base function was provided by the carboxylate group of fatty acids. The participation of the carboxylate group of the substrate was supported by the fact that cytochrome P450_BSβ can catalyze oxidations of nonnatural substrates such as styrene and ethylbenzene in the presence of a series of short-alkyl-chain carboxylic acids as a dummy molecule of fatty acid. We refer to a series of short-alkyl-chain carboxylic acids as a “decoy molecule”. As shown here, we have clarified the crystal structure of the decoy-molecule-bound form and elucidated that the location of its carboxylate group is virtually the same as that of palmitic acid in the heme cavity, indicating that the carboxylate group of the decoy molecule serves as the general acid–base catalyst. This result further confirms that the role of the acid–base function is satisfied by the carboxylate group of the substrates. In addition, the structure analysis of the substrate-free form has clarified that no remarkable structural change is induced by the binding of the decoy molecule as well as fatty acid. Consequently, whether the carboxylate group is positioned in the active site provides the switching mechanism of the catalytic cycle of cytochrome P450_BSβ.     

Engineering of Candida antarctica lipase B for hydrolysis of bulky carboxylic acid esters

Candida antarctica lipase B (CALB) is a widely used biocatalyst with high activity and specificity for a wide range of primary and secondary alcohols. However, the range of converted carboxylic acids is more narrow and mainly limited to unbranched fatty acids. To further broaden the biotechnological applications of CALB it is of interest to expand the range of converted carboxylic acid and extend it to carboxylic acids that are branched or substituted in close proximity of the carboxyl group. An in silico library of 2400 CALB variants was built and screened in silico by substrate-imprinted docking, a four step docking procedure. First, reaction intermediates of putative substrates are covalently docked into enzyme active sites. Second, the geometry of the resulting enzyme-substrate complex is optimized. Third, the substrate is removed from the complex and then docked again into the optimized structure. Fourth, the resulting substrate poses are rated by geometric filter criteria as productive or non-productive poses. Eleven enzyme variants resulting from the in silico screening were expressed in Escherichia coli BL21 and measured in the hydrolysis of two branched fatty acid esters, isononanoic acid ethyl ester and 2-ethyl hexanoic acid ethyl esters. Five variants showed an initial increase in activity. The variant with the highest wet mass activity (T138S) was purified and further characterized. It showed a 5-fold increase in hydrolysis of isononanoic acid ethyl ester, but not toward sterically more demanding 2-ethyl hexanoic acid ethyl ester.     

Aspartic proteinases: Fourier transform infrared spectroscopic studies of a model of the active side.

We synthesized and studied by Fourier transform infrared spectroscopy nine monosalts of diamides as models for the active side of aspartic proteinases. One compound, the monosalt of meta-aminobenzoic acid diamide of fumaric acid (m-FUM), shows the same biological activity as pepsin with regard to the splitting of peptide bonds of the Pro-Thi-Glu-Phe-Phe(4-NO2)-Arg-Leu heptapeptide. The monosalt of m-FUM forms with oxindole a complex in which the carboxylic acid group of the monosalt of m-FUM is strongly hydrogen bonded with the O atom of the peptide bond of oxindole. When one water molecule is added to this complex, the strong field of the carboxylate group destabilizes an O-H bond of the water molecule. The distorted water molecule attacks the carbon atom of the peptide group, and the water proton transfers to the peptide N atom. Simultaneously, the C-N bond of the amide group is broken. Hence it is demonstrated that the catalytic mechanism of aspartic acid proteinases is a base catalysis. The results show that for this catalytic mechanism there are sufficient carboxylic and carboxylate groups, as well as a water molecule in the correct arrangement. It was also demonstrated with other monosalts of dicarboxylic acids that well-defined steric conditions of the carboxylic acid and the carboxylate group must be fulfilled to show hydrolytic activity with regard to oxindole molecules.     

Dicarboxylic acids with limited numbers of hydrocarbons stabilize cell membrane and increase osmotic resistance in rat erythrocytes

We examined the effect of dicarboxylic acids having 0 to 6 hydrocarbons and their corresponding monocarboxylic or tricarboxylic acids in changing the osmotic fragility (OF) in rat red blood cells (RBCs). Malonic, succinic, glutaric and adipic acids, which are dicarboxylic acids with 1, 2, 3 and 4 straight hydrocarbons located between two carboxylic groups, decreased the OF in a concentration-dependent manner. Other long-chain dicarboxylic acids did not change the OF in rat RBCs. The benzoic acid derivatives, isophthalic and terephthalic acids, but not phthalic acid, decreased the OF in a concentration-dependent manner. Benzene-1,2,3-tricarboxylic acid, but not benzene-1,3,5-tricarboxylic acid, also decreased the OF in rat RBCs. On the other hand, monocarboxylic acids possessing 2 to 7 straight hydrocarbons and benzoic acid increased the OF in rat RBCs. In short-chain dicarboxylic acids, a limited number of hydrocarbons between the two carboxylic groups are thought to form a V- or U-shaped structure and interact with phospholipids in the RBC membrane. In benzene dicarboxylic and tricarboxylic acids, a part of benzene nucleus between the two carboxylic groups is thought to enter the plasma membrane and act on acyl-chain in phospholipids in the RBC membrane. For dicarboxylic and tricarboxylic acids, limited numbers of hydrocarbons in molecules are speculated to enter the RBC membrane with the hydrophilic carboxylic groups remaining outside, stabilizing the structure of the cell membrane and resulting in an increase in osmotic resistance in rat RBCs.     

Characterization of acid catalytic domains for cellulose hydrolysis and glucose degradation

Cellulolytic enzymes consist of a catalytic domain, a linking peptide, and a binding domain. The paper describes research on carboxylic acids that have potential as catalytic domains for constructing organic macromolecules for use in cellulose hydrolysis that mimic the action of enzymes. The tested domains consist of the series of mono-, di-, and tricarboxylic acids with a range of pK(a)'s. This paper systematically characterizes the acids with respect to hydrolysis of cellobiose, cellulose in biomass, and degradation of glucose and compares these kinetics data to dilute sulfuric acid. Results show that acid catalyzed hydrolysis is proportional to H+ concentration. The tested carboxylic acids did not catalyze the degradation of glucose while sulfuric acid catalyzed the degradation of glucose above that of water alone. Consequently, overall yields of glucose obtained from cellobiose and cellulose are higher for the best carboxylic acid tested, maleic acid, when compared to sulfuric acid at equivalent solution pH.     

Mechanisms of Carboxylic Acid Attraction in Drosophila melanogaster

Sour is one of the fundamental taste modalities that enable taste perception in animals. Chemoreceptors embedded in taste organs are pivotal to discriminate between different chemicals to ensure survival. Animals generally prefer slightly acidic food and avoid highly acidic alternatives. We recently proposed that all acids are aversive at high concentrations, a response that is mediated by low pH as well as specific anions in Drosophila melanogaster. Particularly, some carboxylic acids such as glycolic acid, citric acid, and lactic acid are highly attractive to Drosophila compared with acetic acid. The present study determined that attractive carboxylic acids were mediated by broadly expressed Ir25a and Ir76b, as demonstrated by a candidate mutant library screen. The mutant deficits were completely recovered via wild-type cDNA expression in sweet-sensing gustatory receptor neurons. Furthermore, sweet gustatory receptors such as Gr5a, Gr61a, and Gr64a-f modulate attractive responses. These genetic defects were confirmed using binary food choice assays as well as electrophysiology in the labellum. Taken together, our findings demonstrate that at least two different kinds of receptors are required to discriminate attractive carboxylic acids from other acids.     

Mechanism and specificity of succinyl-CoA:3-ketoacid coenzyme A transferase.

(a) The reactivity of substituted acetates as substrates for CoA transferase increases sharply with increasing basicity and exhibits a slope of 1.0 in a plot of either log kappacat or log (kappacat/Km) against pKa (betanuc = 1.0). This result shows that the catalyzed reaction, which involves both carboxylate activation and leaving group transfer, does not proceed through a fully concerted reaction mechanism in the rate-determining step. The result is consistent with a stepwise reaction mechanism that proceeds through an anhydride intermediate. (b) Equilibrium constants for thiol ester formation, either bound to the enzyme or free in solution, show the same dependence on the basicity of carboxylate ions (betaeq = 1.0) and are independent of acidity when expressed in terms of the carboxylic acid. Thus, the polar environment around substituents on the acyl group is the same for carboxylic acids, thiol esters, and oxygen esters. (c) The interaction of the terminal CH3CO group of acetoacetate with the active site causes a 200,000-fold increase in kappacat/Km, corresponding to a decrease in delta G++ OF 7.2 kcal/mol compared with an unsubstituted acid of the same pK. The binding energy of the coenzyme A moiety of the substrate is utilized to interact with the active site and cause a 10(4) to 10(6)-fold increase in kappacat, corresponding to a decrease in delta G++ of 6 to 9 kcal/mol, compared with fragments of the coenzyme A moiety added separatly or together. (d) The exchange of labeled coenzyme A into acyl-CoA substrates was found to be greater than or equal to 10(5) slower than substrate turnover.     

Synthesis and characterization of pyridine N-oxide complexes of manganese, copper and zinc

A series of metal carboxylates containing pyridine N-oxide are prepared via one pot synthesis and solid phase synthesis. The structural variations from metal to metal are observed. In the case of reactions of manganese(II) acetate with pyridine N-oxide in the presence of aromatic carboxylic acids, polymeric complexes with bridging aromatic carboxylate as well as bridging pyridine N-oxide are observed. Whereas, the reaction of copper(II) acetate with pyridine N-oxide in the presence of an aromatic carboxylic acid led to mononuclear or binuclear paddle wheel carboxylate complexes with monodentate pyridine N-oxide. Co-crystal of two neutral complexes having composition [Cu₂(OBz)₄(MeOH)₂][Cu₂(OBz)₄(pyO)₂] (where OBz = benzoate, pyO = pyridine N-oxide) each neutral parts have paddle wheel structure. Solid phase reaction of zinc chloride with sodium benzoate prepared in situ and pyridine N-oxide leads to a tetra-nuclear zinc complex.     

Polymer-bound alkyltriazenes for mild racemization-free esterification of amino acid and peptide derivatives.

A novel tool for polymer-assisted solution phase (PASP) esterification of amino acid and peptide derivatives has been developed. When treated with carboxylic acids, polymer-bound alkyltriazenes react with a loss of nitrogen and transfer of the alkyl moiety to the carboxylate anion to form the corresponding alkyl esters. There are no limitations with regard to either the protecting groups or the nature of the amino acid. Furthermore no racemization occurs at the chiral centers of the amino acids as demonstrated by chiral GC-MS analyses. Alkyltriazene-resins were also applied successfully to the esterification of peptide acids and other peptidic structures, such as tripalmitoyl-S-glyceryl-cysteine (Pam3Cys). The triazene-mediated esterification reaction is exceptionally mild, and there is no need for prior activation of the carboxy groups. This method is therefore particularly suitable for the alkylation of complex peptidomimetic structures prone to racemization and for acid-sensitive structures.     

A reinvestigation of the synthesis of arsonolipids (2,3-diacyloxypropylarsonic acids)

A reinvestigation of the reactions leading to arsonolipids (2,3-diacyloxypropylarsonic acids) has been carried out in order to understand why the yields of their preparation were only moderate, although they are better than those reported for 2,3-diacyloxypropylphosphonic acid (phosphotidic acid). Thus, the reaction of glycidol and of 3-chloro-1,2-propanediol with alkaline sodium arsenite, "Na3AsO3", gives the desired product, 2,3-dihydroxypropylarsonic acid, and approximately 10% of an arsenic-containing glycerol dimer which is removed during the preparation of these arsonolipids. The step which is mainly responsible for the diminished yields is due to the reaction of the -As(SPh)2 or -AsO3H- precursor with the activated acid chlorides or carboxylic acid anhydrides to give an intermediate which cyclizes with the primary hydroxy group of the 2,3-dihydroxypropyl moiety. This cyclization does not allow the primary hydroxy group to be acylated. Such cyclization could not be avoided with RCOCl/py, (RCO)2O/DMAP, or RCOOH/DCC/DMAP acylating systems.     

An Efficient Conversion of Carboxylic Acids to One-Carbon Degraded Aldehydes via 2-Hydroperoxy Acids

After the formation of dianions of a carboxylic acid with lithium diisopropylamide, oxygen was bubbled into the solution to produce 2-hydroperoxy acid. Then the reaction mixture was acidified with a 2 N HCl solution and subsequently elevated to 50 °C to afford the aldehyde with the loss of one carbon atom. Even saturated (C₁₀–C₂₀) and unsaturated (C_18:1) carboxylic acids were converted into the odd aldehydes (C₉–C₁₉, C_17:1) in high yields. This conversion was found to be an efficient method for the preparation of carboxylic acids (Cn) to one-carbon degraded aldehydes (Cn-1) via 2-hydroperoxy acids.     

The Aldehyde Fuchsin and colloidal iron staining reactions in the canine thyroid C cell

Synopsis The cytochemically reactive groups which are responsible for Aldehyde Fuchsin (AF) and colloidal iron (CI) staining of C cells were investigated in the canine thyroid gland. To this end, stains for proteoglycans and peptide groups were utilized in conjuction with hydrolysis of glycosidic and amide bonds. In addition, the following procedures were used: acetylation, benzoylation, nitrozation, aldehyde blockade, sulphydryl blockade, methylation and mild acid hydrolysis.No acidic proteoglycan, sialic acid, polyphosphate or polysaccharide ester sulphate were detected in C cells; the results suggest that AF staining, after an oxidation step, and CI staining are due to polypeptides. Sulphydryl and carboxyl groups together are necessary for mediating the attachment of AF in C cells and it is adduced that this attachment is due to the combined charges of sulphonic and carboxylic acids. Methylation and acetylation inhibit CI staining and those staining reactions that depend upon carboxylic acid (TB) and hydroxyl groups (PAS) for their dye attachment in C cells. acid hydrolysis, which increases the demonstration of carboxylic acid in C cells, decreases the attachment of hydroxyferric ions. I speculate that this inhibition is due to extraction of iron binding sites in the C cell and conclude that it is not solely carboxylic acids in C cells that are responsible for CI staining.     

A chemoenzymatic synthesis of deoxy sugar esters involving stereoselective acetylation of hemiacetals catalyzed by CALB

An extension of the scope of the chemoenzymatic strategy for the synthesis of stereochemically pure pyranose deoxy sugar esters of different carboxylic acids has been achieved. The objective of the work was to extend the strategy to the synthesis of furanose deoxy sugar derivatives and additionally, to N-Boc-protected amino acid esters. With all used carboxylic acids (deoxycholic acid, α-methoxyphenylacetic acid, N-Boc-l-phenylalanine and N-Boc-l-tyrosine) the lipase-catalyzed stereoselective acetylation of furanose or pyranose hemiacetal moiety as a key step afforded one desired stereochemically pure acetylated hemiacetal deoxy sugar ester in high de.     

Interaction of metal ions with carboxylic and carboxamide groups in protein structures

An analysis of the geometry of metal binding by carboxylic and carboxamide groups in proteins is presented. Most of the ligands are from aspartic and glutamic acid side chains. Water molecules bound to carboxylate anions are known to interact with oxygen lone-pairs. However, metal ions are also found to approach the carboxylate group along the C-O direction. More metal ions are found to be along the syn than the anti lone-pair direction. This seems to be the result of the stability of the five-membered ring that is formed by the carboxylate anion hydrogen bonded to a ligand water molecule and the metal ion in the syn position. Ligand residues are usually from the helix, turn or regions with no regular secondary structure. Because of the steric interactions associated with bringing all the ligands around a metal center, a calcium ion can bind only near the ends of a helix; a metal, like zinc, with a low coordination number, can bind anywhere in the helix. Based on the analysis of the positions of water molecules in the metal coordination sphere, the sequence of the EF hand (a calcium-binding structure) is discussed.     

Emission spectra from copper proteins containing type-3 centres

Aqueous solutions of copper-proteins containing type-3 centres (ceruloplasmin, tyrosinase, haemocyanin), excited within their absorption bands at 325-345 nm, show typical luminescence spectra. The emission bands peak at 415-445 nm and their decay time is no longer than 10 ns. A strong analogous fluorescence is obtained also by excitation of concentrated solutions of carboxylic acids and amino acids, which show again absorption bands around 330 nm. Such a fluorescence, although less intense, is also observed in copper(II) carboxylate solutions. In contrast, no fluorescence has been recorded in solutions of acetic anhydride and of polypeptides (valinomycin, gramicidin D), which do not have free carboxyl groups. We tentatively attribute this novel fluorescence in the investigated copper proteins to interactions between carboxyl groups of amino acids at, or near, the active site.